Expression cassettes for regulation of expression in monocotyledonous plants

ABSTRACT

The present invention relates to expression cassettes comprising at least one transcription regulating nucleotide sequence obtainable from the group of genes of monocotyledonous plants consisting of caffeoyl-CoA-O-methyltransferase genes, C8,7-sterol isomerase genes, hydroxyproline-rich glycoprotein (HRGP) genes, lactate dehydrogenase genes, and chloroplast protein 12 like genes. More preferably the transcription regulating sequences are obtainable from  Zea mays  or  Oryza sativa . The transcription regulating sequences are especially useful for root/kernel-preferential, leaf/endosperm-preferential, root/silk/kernel-preferential, or constitutive expression.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/883,996 filed Aug. 8, 2007, which is a national stage application(under 35 U.S.C. 371) of PCT/EP2006/050781 filed Feb. 8, 2006, whichclaims benefit of U.S. application 60/651,268 filed Feb. 9, 2005. Theentire contents of each of these applications are hereby incorporated byreference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_Listing_(—)074021_(—)0064_(—)01. Thesize of the text file is 175 KB, and the text file was created on Oct.3, 2014.

FIELD OF THE INVENTION

The present invention relates to expression cassettes comprising atleast one transcription regulating nucleotide sequence obtainable fromthe group of genes of monocotyledonous plants consisting ofcaffeoyl-CoA-O-methyltransferase genes, C8,7-sterol isomerase genes,hydroxyproline-rich glycoprotein (HRGP) genes, lactate dehydrogenasegenes, and chloroplast protein 12 like genes. More preferably thetranscription regulating sequences are obtainable from Zea mays or Oryzasativa. The transcription regulating sequences are especially useful forroot/kernel-preferential, leaf/endosperm-preferential,root/silk/kernel-preferential, or constitutive expression.

BACKGROUND OF THE INVENTION

Manipulation of plants to alter and/or improve phenotypiccharacteristics (such as productivity or quality) requires theexpression of heterologous genes in plant tissues. Such geneticmanipulation relies on the availability of a means to drive and tocontrol gene expression as required. For example, genetic manipulationrelies on the availability and use of suitable promoters which areeffective in plants and which regulate gene expression so as to give thedesired effect(s) in the transgenic plant.

Constitutive promoters are favored in situations where expression in all(or most) tissues during all (or most) times of the plant development isrequired. The number of constitutive promoters functional inmonocotyledonous plants is limited and include the rice actin 1 (Wang1992; U.S. Pat. No. 5,641,876), CaMV 35S (Odell 1985), CaMV 19S (Lawton1987), and the maize ubiquitin promoters (Christensen 1996). Whileseveral constitutive and tissue-specific promoters from dicotyledonousplants are described by sequence (e.g., the promoter from thecaffeoyl-CoA-O-methyltransferase gene from parsley (Grimmig 1997),poplar (Chen 1998) and pine (Li 1999)) only a very limited number hasbeen characterized in heterogenous gene expression. In comparison withdicotyledonous promoters, promoters from monocotyledonous plants arestill very limited. It is advantageous to have the choice of a varietyof different promoters so that the most suitable promoter may beselected for a particular gene, construct, cell, tissue, plant, orenvironment. Moreover, the increasing interest in cotransforming plantswith multiple plant transcription units (PTU) and the potential problemsassociated with using common regulatory sequences for these purposesmerit having a variety of promoter sequences available.

Root-preferential or root-specific promoters are useful for alterationof the function of root tissue, modification of growth rate, improvementof resistance to root preferred pathogens, pests, herbicides or adverseweather conditions, for detoxification of soil as well as for broadeningthe range of soils or environments in which said plant may grow. Rootabundant or root specific gene expression would provide a mechanismaccording to which morphology and metabolism may be altered to improvethe yield and to produce useful proteins in greater amounts. Inparticular, root specific promoters may be useful for expressingdefense-related genes, including those conferring insectical resistanceand stress tolerance, e.g. salt, cold or drought tolerance, and genesfor altering nutrient uptake. The number of root preferential androot-specific promoters functional in monocotyledonous plants is verylimited. These include the MR7 promoter from Zea mays (U.S. Pat. No.5,837,848), the ZRP2 promoter of Zea mays (U.S. Pat. No. 5,633,363), andthe MTL promoter from Zea mays (U.S. Pat. Nos. 5,466,785 and 6,018,099).Many of these examples disclose promoters with expression patternsconfined to a limited number of root tissues. Other fail to provide theroot specificity needed for expression of selected genes. It isadvantageous to have the choice of a variety of different promoters sothat the most suitable promoter may be selected for a particular gene,construct, cell, tissue, plant, or environment. Moreover, the increasinginterest in cotransforming plants with multiple plant transcriptionunits (PTU) and the potential problems associated with using commonregulatory sequences for these purposes merit having a variety ofpromoter sequences available.

There is, therefore, a great need in the art for the identification ofnovel sequences that can be used for expression of selected transgenesin the economically most important monocotyledonous plants, especiallyin rice and maize. It is thus an objective of the present invention toprovide new and alternative expression cassettes for expression oftransgenes in monocotyledonous plants, more preferably with theopportunity to modulate the tissue specificity of expression

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Map of Os.CCoAMT1 promoter::Zm.ubiquitin intron::GUS(PIV2)::CCoAMT1 terminator chimeric construct (pBPSMM325).

-   -   The plasmid comprises an expression construct containing an        Os.CCoAMT1 promoter operably linked to Zm.ubiquitin intron, a        β-glucuronidase gene (GUS including the potato invertase [PIV]2        intron), and 3′ untranslated region and transcriptional        termination region of the Os.CCoAMT1. SM cassette is        representing a selection marker (ahas) cassette.

FIG. 2 GUS expression controlled by Oryza sativa (Os) CCoAMT1 promoterconstruct (pBPSMM325) in maize. The upper panel (I) represents theoriginal photos with the GUS staining, while the lower panel (II)indicates areas distinctly stained blue by overlaid shaded areas.

-   -   (A) Leaf+root at the 5 leaf stage    -   (B) Leaf at flowering stage    -   (C) Kernel (prepollination)    -   (D) Kernel 30 DAP    -   Pictures represent reproducible expression patterns from 15 T₁        single copy lines.

FIG. 3 A: Map of Os.CCoAMT1 promoter::Zm.ubiquitin intron::GUS(PIV2)::NOS terminator fusion construct (pBPSMM271).

-   -   The plasmid comprises an expression construct containing an        Os.CCoAMT1 promoter operably linked to Zm.ubiquitin intron, a        β-glucuronidase gene (GUS including the potato invertase [PIV]2        intron), and nopaline synthase (NOS) terminator. The SM cassette        is representing a selection marker (ahas) cassette.    -   B: GUS expression controlled by Os.CCoAMT1 promoter construct        (pBPSMM271) in maize. The upper panel (I) represents the        original photos with the GUS staining, while the lower        panel (II) indicates areas distinctly stained blue by overlaid        shaded areas.    -   (A) Leaves and roots at the 5 leaf stage    -   (B) Leaf at the flowering stage    -   (C) Kernel (30 d after pollination: DAP)    -   Pictures represent reproducible expression patterns from 15 T₁        single copy lines.

FIG. 4 A: Map of Os.SI::Zm.ubiquitin intron::GUS (PIV2)::NOS terminatorfusion construct (pBPSMM331).

-   -   The plasmid comprises an expression construct containing an        Os.SI promoter operably linked to Zm.ubiquitin intron, a        β-glucuronidase gene (GUS including the potato invertase [PIV]2        intron), and NOS terminator. The SM cassette is representing a        selection marker cassette.    -   B: GUS expression controlled by Os.SI promoter construct        (pBPSMM331) in maize. The upper panel (I) represents the        original photos with the GUS staining, while the lower        panel (II) indicates areas distinctly stained blue by overlaid        shaded areas.    -   (A) Leaves and roots at the 5 leaf stage    -   (B) Leaf at the 5 leaf stage Leaf at the flowering stage    -   (C) Kernel (30 d after pollination: DAP)    -   Pictures represent reproducible expression patterns from 15 T₁        single copy lines.

FIG. 5 A: Map of Zm.HRGP::Zm.ubiquitin intron::GUS (PIV2)::Zm.HRGPterminator fusion construct (pBPSET003).

-   -   The plasmid comprises an expression construct containing a        Zm.HRGP promoter operably linked to Zm.ubiquitin intron, a        β-glucuronidase gene (GUS including the potato invertase [PIV]2        intron), and HRGP terminator. The SM cassette is representing a        selection marker (ahas) cassette.    -   B: GUS expression controlled by maize Zm.HRGP promoter construct        (pBPSET003) in maize. The upper panel (I) represents the        original photos with the GUS staining, while the lower        panel (II) indicates areas distinctly stained blue by overlaid        shaded areas.    -   (A) Leaves and roots at the 5 leaf stage    -   (B) Leaf at the 5 leaf stage Leaf at the flowering stage    -   (C) Kernel (30 d after pollination: DAP)    -   Pictures represent reproducible expression patterns from 15 T₁        single copy lines.

FIG. 6 A: Map of Zm.LDH::Zm.ubiquitin intron::GUS (PIV2)::NOS terminatorfusion construct (pBPSMM272).

-   -   The plasmid comprises an expression construct containing a        Zm.LDH promoter operably linked to Zm.ubiquitin intron, a        β-glucuronidase gene (GUS including the potato invertase [PIV]2        intron), and NOS terminator. The SM cassette is representing a        selection marker (ahas) cassette.    -   B: GUS expression controlled by maize Zm.LDH promoter construct        (pBPSMM272) in maize. The upper panel (I) represents the        original photos with the GUS staining, while the lower        panel (II) indicates areas distinctly stained blue by overlaid        shaded areas. The transgenic plants containing either pBPSMM272        or pBPSET007 showed the same expression patterns.    -   (A) Leaves and roots at the 5 leaf stage    -   (B) Leaf at the 5 leaf stage Leaf at the flowering stage    -   (C) Kernel (30 d after pollination: DAP)    -   Pictures represent reproducible expression patterns from 8 T₁        single copy lines.

FIG. 7 A: Map of Zm.LDH::Zm.ubiquitin intron::GUS (PIV2)::Zm.LDHterminator fusion construct (pBPSET007).

-   -   The plasmid comprises an expression construct containing a        Zm.LDH promoter operably linked to Zm.ubiquitin intron, a        β-glucuronidase gene (GUS including the potato invertase [PIV]2        intron), and LDH terminator. The SM cassette is representing a        selection marker (ahas) cassette.    -   B: GUS expression controlled by maize Zm.LDH promoter construct        (pBPSET007) in maize. The upper panel (I) represents the        original photos with the GUS staining, while the lower        panel (II) indicates areas distinctly stained blue by overlaid        shaded areas.    -   (A) Leaves and roots at the 5 leaf stage    -   (B) Leaf at the flowering stage    -   (C) Kernel (30 d after pollination: DAP)    -   Pictures represent reproducible expression patterns from 15 T₁        single copy lines.

FIG. 8 A: Map of Os.CP12::Zm.ubiquitin intron::GUS (PIV2)::NOSterminator fusion construct (pBPSMM304).

-   -   The plasmid comprises an expression construct containing a        Os.CP12 promoter operably linked to Zm.ubiquitin intron, a        β-glucuronidase gene (GUS including the potato invertase [PIV]2        intron), and NOS terminator. SM cassette is representing a        selection marker cassette.    -   B: GUS expression controlled by maize Os.CP12 promoter construct        (pBPSMM304) in maize. The upper panel (I) represents the        original photos with the GUS staining, while the lower        panel (II) indicates areas distinctly stained blue by overlaid        shaded areas.    -   (A) Leaves and roots at the 5 leaf stage    -   (B) Leaf at the flowering stage    -   (C) Kernel (30 d after pollination: DAP)    -   Pictures represent reproducible expression patterns from 15 T₁        single copy lines.

FIG. 9 Drought-stress-induced expression of Zm.LDH promoter construct(pBPSMM272) in maize. Transgenic plants at 5-leaf stage weredrought-stressed by withholding water. Samples were taken from leaves atthe indicated time points. RNA was isolated from leaf samples andanalyzed with quantitative RT-PCR. GUS expression was normalized againstan internal control gene in each sample. Results are shown as foldincrease of expression levels compared to the 0-timepoint, which is setas 1.

FIG. 10A-B Protein alignment of rice lactate dehydrogenase (LDH) proteinwith the LDH proteins from maize (1) (SEQ ID NO: 26), rice (2) (SEQ IDNO: 65), barley (3) (SEQ ID NO: 95), rice (4) (SEQ ID NO: 60),Arabidopsis (5, 6) (SEQ ID NO: 96 and SEQ ID NO: 97), tomato (7) (SEQ IDNO: 98), potato (8) (SEQ ID NO: 99).

-   -   The sequences motifs distinguishing monocotyledonous LDH        proteins from other dicotyledonous LDH proteins are boxed        (relevant different amino acids are marked with a “+”). Further        such sequences motifs may be readily identified by the person        skilled in the art based on the present alignment.

FIG. 11. Protein alignment of rice C8,7 sterol isomerase (SI) proteinwith the SI proteins from Arabidopsis (1-3) (SEQ ID NO: 100, SEQ ID NO:101, SEQ ID NO: 102) and rice (4) (SEQ ID NO: 10).

FIG. 12. Protein alignment of rice Caffeoyl CoA-O-methyltransferase 1(CCoAMT1) with the CCoAMT1 proteins from tobacco (1) (SEQ ID NO: 103),eucalyptus (2) (SEQ ID NO: 104), popular (3) (SEQ ID NO: 105), maize (4,5, 6) (SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 70) and rice (7) (SEQID NO: 5). The sequences motifs distinguishing monocotyledonous CCoAMTproteins from other dicotyledonous CCoAMT proteins are boxed (relevantdifferent amino acids are marked with a “+”). Further such sequencesmotifs may be readily identified by the person skilled in the art basedon the present alignment.

SUMMARY OF THE INVENTION

Accordingly, a first embodiment of the invention relates to expressioncassettes for regulating expression in monocotyledonous plantscomprising

-   i) at least one transcription regulating nucleotide sequence of a    monocotyledonous plant gene, said monocotyledonous plant gene    selected from the group of genes consisting of    caffeoyl-CoA-O-methyltransferase genes, C8,7-sterol isomerase genes,    hydroxyproline-rich glycoprotein (HRGP) genes, lactate dehydrogenase    genes, and chloroplast protein like 12 genes, and functionally    linked thereto-   ii) at least one nucleic acid sequence which is heterologous in    relation to said transcription regulating sequence.

Preferably, the transcription regulating nucleotide sequence isobtainable from monocotyledonous plant genomic DNA from a gene encodinga polypeptide which

-   a1) comprises at least one sequence motif of a monocotyledonous    plant lactate dehydrogenase protein selected from the group    consisting of the amino acid sequences

i) SLSELGFDA, (SEQ ID NO: 76) ii) VIGAGNVGMA, (SEQ ID NO: 77) iii)IVTAGARQI, (SEQ ID NO: 78) iv) L(F/Y)RKIVP, (SEQ ID NO: 79) v) GFPASRV,(SEQ ID NO: 80) vi) RF(L/I)AEHL, (SEQ ID NO: 81) vii) QAYMVGEH,(SEQ ID NO: 82) viii) ALEGIRRAV, (SEQ ID NO: 83) and ix) GYSVAS(L/I)A,(SEQ ID NO: 84)

-   -   or

-   b1) is encoding a lactate dehydrogenase protein from a    monocotyledonous plant having an amino acid sequence identity of at    least 90% to a polypeptide selected from the group described by SEQ    ID NO: 26, 60 and 65, or

-   a2) comprises at least one sequence motif of a monocotyledonous    plant caffeoyl-CaA-O-methyltransferase protein selected from the    group consisting of the amino acid sequences

x) EQKTRHSE, (SEQ ID NO: 85) xi) L(I/L)KLIGAK, (SEQ ID NO: 86) xii)KTMEIGVY, (SEQ ID NO: 87) xiii) HERL(L/M)KLV, (SEQ ID NO: 88) xiv)CQLPVGDG, (SEQ ID NO: 89) and xv) TLCRRVK, (SEQ ID NO: 90)

-   -   or

-   b2) is encoding a caffeoyl-CaA-O-methyltransferase protein from a    monocotyledonous plant having an amino acid sequence identity of at    least 90% to a polypeptide selected from the group described by SEQ    ID NO: 5, and 70, or

-   b3) is encoding a hydroxyproline-rich glycoprotein from a    monocotyledonous plant having an amino acid sequence identity of at    least 90% to a polypeptide selected from the group described by SEQ    ID NO: 18, and 75, or

-   b4) is encoding a C-8,7-stereol-isomerase protein from a    monocotyledonous plant having an amino acid sequence identity of at    least 90% to a polypeptide selected described by SEQ ID NO: 10, or

-   b5) is encoding a Chloroplast protein 12 like protein from a    monocotyledonous plant having an amino acid sequence identity of at    least 90% to a polypeptide described by SEQ ID NO: 31.

Preferably, the transcription regulating nucleotide sequence is from acorn (Zea mays) or rice (Oryza sativa) plant. Even more preferably thetranscription regulating nucleotide sequence is from a plant geneselected from the group of genes consisting of Oryza sativacaffeoyl-CoA-O-methyltransferase genes, Oryza sativa C8,7-sterolisomerase genes, Zea may hydroxyproline-rich glycoprotein (HRGP) genes,Zea mays lactate dehydrogenase genes, Oryza sativa chloroplast protein12 like genes and functional equivalents thereof. The functionalequivalent gene is preferably encoding a polypeptide which has at least90% amino acid sequence identity to a polypeptide selected from thegroup described by SEQ ID NO: 5, 10, 18, 26, 31, 60, 65, 70, and 75.

In a more preferred embodiment the transcription regulating nucleotidesequence is selected from the group of sequences consisting of

-   i) the sequences described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12,    13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61,    62, 63, 66, 67, 68, 71, 72, and 73, and-   ii) a fragment of at least 50 consecutive bases of a sequence under    i); and-   iii) a nucleotide sequence having substantial similarity (preferably    with a sequence identity of at least 60%; more preferably measured    by the BLASTN program with the default parameters wordlength (W) of    11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a    comparison of both strands) to a transcription regulating nucleotide    sequence described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14,    15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63,    66, 67, 68, 71, 72, or 73; and-   iv) a nucleotide sequence capable of hybridizing to a transcription    regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 6,    7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29,    56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73 or the complement    thereof; and-   v) a nucleotide sequence capable of hybridizing to a nucleic acid    comprising 50 to 200 or more consecutive nucleotides of a    transcription regulating nucleotide sequence described by SEQ ID NO:    1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24,    27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73 or the    complement thereof (preferably in 7% sodium dodecyl sulfate (SDS),    0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at    50° C.; more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M    NaPO₄, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50°    C., still more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M    NaPO₄, 1 mM EDTA at 50° C. with washing in 0.5×SSC, 0.1% SDS at 50°    C., even more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M    NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50°    C., most preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄,    1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.);    and-   vi) a nucleotide sequence which is the complement or reverse    complement of any of the previously mentioned nucleotide sequences    under i) to v).

Another preferred embodiment relates to an expression cassette forregulating expression in monocotyledonous plants comprising

-   a) at least one transcription regulating nucleotide sequence    functional in a monocotyledonous plant comprising at least one    sequence selected from the group of sequences consisting of    -   i) the sequences described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11,        12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57,        58, 61, 62, 63, 66, 67, 68, 71, 72, and 73, and    -   ii) a fragment of at least 50 consecutive bases of a sequence        under i); and    -   iii) a nucleotide sequence having substantial similarity        (preferably with a sequence identity of at least 60%; more        preferably measured by the BLASTN program with the default        parameters wordlength (W) of 11, an expectation (E) of 10, a        cutoff of 100, M=5, N=−4, and a comparison of both strands) to a        transcription regulating nucleotide sequence described by SEQ ID        NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22,        23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72,        or 73; and    -   iv) a nucleotide sequence capable of hybridizing to a        transcription regulating nucleotide sequence described by SEQ ID        NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22,        23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72,        or 73 or the complement thereof (preferably in 7% sodium dodecyl        sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in        2×SSC, 0.1% SDS at 50° C.; more preferably in 7% sodium dodecyl        sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in        1×SSC, 0.1% SDS at 50° C., still more preferably in 7% sodium        dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with        washing in 0.5×SSC, 0.1% SDS at 50° C., even more preferably in        7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at        50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., most        preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM        EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.); and    -   v) a nucleotide sequence capable of hybridizing to a nucleic        acid comprising 50 to 200 or more consecutive nucleotides of a        transcription regulating nucleotide sequence described by SEQ ID        NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22,        23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72,        or 73 or the complement thereof; and    -   vi) a nucleotide sequence which is the complement or reverse        complement of any of the previously mentioned nucleotide        sequences under i) to v),    -   and-   b) at least one nucleic acid sequence which is heterologous in    relation to said transcription regulating sequence.

Preferably, the sequences specified under ii), iii), iv) v) and vi) inthe paragraphs above are capable to modify transcription in amonocotyledonous plant cell or organism. More preferably said sequencesspecified under ii), iii), iv) v) and vi) have substantially the sametranscription regulating activity as the transcription regulatingnucleotide sequence described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12,13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62,63, 66, 67, 68, 71, 72, or 73.

Also preferably the sequences specified under iii) above have a sequenceidentity of at least 60%, preferably 70% or 80%, more preferably 90% or95% to a sequence described by SEQ ID NO: 1, 2, 3, 6, 7, 8, 11, 12, 13,14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63,66, 67, 68, 71, 72, or 73, wherein the identity is preferably measuredby the BLASTN program with the default parameters wordlength (W) of 11,an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparisonof both strands.

Further preferably, the sequences specified under iv) or v) above arehybridizing under stringent conditions, preferably under mediumstringent conditions, most preferably under high stringent conditions(such as in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.) with the specifiedtarget sequence.

In one preferred embodiment of the expression cassette of the invention,the expression of the nucleic acid sequence results in expression of aprotein, or expression of an antisense RNA, sense or double-strandedRNA.

The expression profile of the expression cassettes of the invention maybe modulated depending on the combination of the transcriptionregulating nucleotide sequence with expression enhancing introns and/ortranscriptions termination sequences. This in a preferred embodiment theexpression cassette of the inventions comprises at least one additionalelement selected from the group consisting of

a) 5′-untranslated regions, andb) intron encoding sequences, andc) transcription termination sequences.

The intron encoding sequences are preferably encoding an expressionenhancing intron from a monocotyledonous plant. More preferably theintron sequence is an intron from an ubiquitin, actin or alcoholdehydrogenase gene. Preferably, this intron is inserted in theexpression construct in the 5′-untranslated region of the nucleic acidsequence, which should be expressed (i.e., between the transcriptionregulating nucleotide sequence and the protein coding sequence (openreading frame) or the nucleic acid sequence to be expressed).

Preferably, the 5′-untranslated region is from the same gene as thetranscription regulating sequences.

The transcription terminating sequence preferably also comprises asequence inducing polyadenylation. The transcription terminatingsequence may be heterologous with respect to the transcriptionregulating nucleotide sequence and/or the nucleic acid sequence to beexpressed, but may also be the natural transcription regulatingnucleotide sequence of the gene of said transcription regulatingnucleotide sequence and/or said nucleic acid sequence to be expressed.In one preferred embodiment of the invention the transcriptionregulating nucleotide sequence is the natural transcription regulatingnucleotide sequence of the gene of the transcription regulatingsequence. Preferably the transcription termination sequence is selectedfrom the group of sequences described by SEQ ID NO: 32, 34, and 35.

The transcription regulating sequences of the invention are especiallyuseful for constitutive or root/kernel-preferential orroot/kernel-specific expression in monocotyledonous plants. However, ause in other plants (e.g., dicotyledonous or gymnosperm plants) andother tissues cannot be ruled out. It has been shown that the tissuespecificity of the transcription regulating sequences of the inventioncan be advantageously modulated by the combination with introns and/ortranscription termination sequences.

The expression cassette may be employed for numerous expression purposessuch as for example expression of a protein, or expression of anantisense RNA, sense or double-stranded RNA. Preferably, expression ofthe nucleic acid sequence confers to the plant an agronomically valuabletrait.

Some of the transcription regulating sequences of the invention arenovel even as such (i.e. as isolated nucleotide sequences). Accordinglyanother embodiment of the invention relates to an isolated nucleic acidsequence comprising at least one transcription regulating nucleotidesequence as described by SEQ ID NO: 6, 7, 8, 11, 12, 13, 19, 20, or 21.

Other embodiments of the invention relate to vectors comprising anexpression cassette of the invention, and transgenic host cell ornon-human organism comprising an expression cassette or a vector of theinvention. Preferably the organism is a plant, more preferably amonocotyledonous plant, most preferably selected form the groupconsisting of Zea mays (corn), Oryza sativa (rice), Triticum aestivum(wheat), Hordeum vulgare (barley), and Avena sativa (oats).

Another embodiment of the invention relates to a method for identifyingand/or isolating transcription regulating nucleotide sequence from amonocotyledonous plant characterized that said identification and/orisolation utilizes a nucleic acid sequence encoding an amino acidsequence as described by SEQ ID NOs: 5, 10, 18, 26, 31, 60, 65, 70, or75, or a part of at least 15 bases of said nucleic acid sequence.Preferably the employed nucleic acid sequences is described by SEQ IDNOs: 4, 9, 17, 25, 30, 59, 64, 69, or 74 or a part of at least 15 basesof said nucleic acid sequence. Preferably said identification and/orisolation is realized by a method selected from polymerase chainreaction, hybridization, and database screening.

Still another embodiment of the invention relates to a method forproviding a transgenic expression cassette for heterologous expressionin monocotyledonous plants comprising the steps of:

-   I. isolating of a transcription regulating nucleotide sequence from    a monocotyledonous plant utilizing at least one nucleic acid    sequence or a part thereof, wherein said sequence is encoding a    polypeptide described by SEQ ID NOs: 5, 10, 18, 26, 31, 60, 65, 70,    or 75, or a part of at least 15 bases of said nucleic acid sequence,    and-   II. functionally linking said transcription regulating nucleotide    sequence to another nucleotide sequence of interest, which is    heterologous in relation to said transcription regulating nucleotide    sequence.

For both of the above mentioned methods preferably the nucleotidesequence utilized for isolation of said transcription regulatingnucleotide sequence is encoding a polypeptide comprising

-   a1) at least one sequence motif of a monocotyledonous plant lactate    dehydrogenase protein selected from the group consisting of the    amino acid sequences

i) SLSELGFDA, (SEQ ID NO: 76) ii) VIGAGNVGMA, (SEQ ID NO: 77) iii)IVTAGARQI, (SEQ ID NO: 78) iv) L(F/Y)RKIVP, (SEQ ID NO: 79) v) GFPASRV,(SEQ ID NO: 80) vi) RF(L/I)AEHL, (SEQ ID NO: 81) vii) QAYMVGEH,(SEQ ID NO: 82) viii) ALEGIRRAV, (SEQ ID NO: 83) and ix) GYSVAS(L/I)A,(SEQ ID NO: 84)

-   -   or

-   a2) at least one sequence motif of a monocotyledonous plant    caffeoyl-CaA-O-methyltransferase protein selected from the group    consisting of the amino acid sequences

x) (SEQ ID NO: 85) EQKTRHSE, xi) (SEQ ID NO: 86) L(I/L)KLIGAK, xii)(SEQ ID NO: 87) KTMEIGVY, xiii)  (SEQ ID NO: 88) HERL(L/M)KLV, xiv)(SEQ ID NO: 89) CQLPVGDG,  and xv) (SEQ ID NO: 90) TLCRRVK.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, plant species or genera,constructs, and reagents described as such. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by the appendedclaims. It must be noted that as used herein and in the appended claims,the singular forms “a,” “and,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a vector” is a reference to one or more vectors and includesequivalents thereof known to those skilled in the art, and so forth.

The term “about” is used herein to mean approximately, roughly, around,or in the region of. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20 percent, preferably 10 percent up ordown (higher or lower).

As used herein, the word “or” means any one member of a particular listand also includes any combination of members of that list.

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.For example, gene refers to a nucleic acid fragment that expresses mRNAor functional RNA, or encodes a specific protein, and which includesregulatory sequences. Genes also include non-expressed DNA segmentsthat, for example, form recognition sequences for other proteins. Genescan be obtained from a variety of sources, including cloning from asource of interest or synthesizing from known or predicted sequenceinformation, and may include sequences designed to have desiredparameters.

The term “intron” refers to sections of DNA (intervening sequences)within a gene that do not encode part of the protein that the geneproduces, and that is spliced out of the mRNA that is transcribed fromthe gene before it is exported from the cell nucleus. Intron sequencerefers to the nucleic acid sequence of an intron. Thus, introns arethose regions of DNA sequences that are transcribed along with thecoding sequence (exons) but are removed during the formation of maturemRNA. Introns can be positioned within the actual coding region or ineither the 5′ or 3′ untranslated leaders of the pre-mRNA (unsplicedmRNA). Introns in the primary transcript are excised and the codingsequences are simultaneously and precisely ligated to form the maturemRNA. The junctions of introns and exons form the splice site. Thesequence of an intron begins with GU and ends with AG. Furthermore, inplants, two examples of AU-AC introns have been described: intron 14 ofthe RecA-like protein gene and intron 7 of the G5 gene from Arabidopsisthaliana are AT-AC introns, Pre-mRNAs containing introns have threeshort sequences that are—beside other sequences—essential for the intronto be accurately spliced. These sequences are the 5′ splice-site, the 3′splice-site, and the branchpoint. mRNA splicing is the removal ofintervening sequences (introns) present in primary mRNA transcripts andjoining or ligation of exon sequences. This is also known ascis-splicing which joins two exons on the same RNA with the removal ofthe intervening sequence (intron). The functional elements of an introncomprising sequences that are recognized and bound by the specificprotein components of the spliceosome (e.g. splicing consensus sequencesat the ends of introns). The interaction of the functional elements withthe spliceosome results in the removal of the intron sequence from thepremature mRNA and the rejoining of the exon sequences. Introns havethree short sequences that are essential—although not sufficient—for theintron to be accurately spliced. These sequences are the 5′ splice site,the 3′ splice site and the branchpoint The branchpoint sequence isimportant in splicing and splice-site selection in plants. Thebranchpoint sequence is usually located 10-60 nucleotides upstream ofthe 3′ splice site. Plant sequences exhibit sequence deviations in thebranchpoint, the consensus sequences being CURAY or YURAY.

The term “native” or “wild type” gene refers to a gene that is presentin the genome of an untransformed cell, i.e., a cell not having a knownmutation.

A “marker gene” encodes a selectable or screenable trait.

The term “chimeric gene” refers to any gene that contains

-   1) DNA sequences, including regulatory and coding sequences, that    are not found together in nature, or-   2) sequences encoding parts of proteins not naturally adjoined, or-   3) parts of promoters that are not naturally adjoined.

Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or compriseregulatory sequences and coding sequences derived from the same source,but arranged in a manner different from that found in nature.

A “transgene” refers to a gene that has been introduced into the genomeby transformation and is stably maintained. Transgenes may include, forexample, genes that are either heterologous or homologous to the genesof a particular plant to be transformed. Additionally, transgenes maycomprise native genes inserted into a non-native organism, or chimericgenes. The term “endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism but that is introduced by genetransfer.

An “oligonucleotide” corresponding to a nucleotide sequence of theinvention, e.g., for use in probing or amplification reactions, may beabout 30 or fewer nucleotides in length (e.g., 9, 12, 15, 18, 20, 21 or24, or any number between 9 and 30). Generally specific primers areupwards of 14 nucleotides in length. For optimum specificity and costeffectiveness, primers of 16 to 24 nucleotides in length may bepreferred. Those skilled in the art are well versed in the design ofprimers for use processes such as PCR. If required, probing can be donewith entire restriction fragments of the gene disclosed herein which maybe 100's or even 1,000's of nucleotides in length.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

The nucleotide sequences of the invention can be introduced into anyplant. The genes to be introduced can be conveniently used in expressioncassettes for introduction and expression in any plant of interest. Suchexpression cassettes will comprise the transcriptional initiation regionof the invention linked to a nucleotide sequence of interest. Preferredpromoters include constitutive, tissue-specific, developmental-specific,inducible and/or viral promoters. Such an expression cassette isprovided with a plurality of restriction sites for insertion of the geneof interest to be under the transcriptional regulation of the regulatoryregions. The expression cassette may additionally contain selectablemarker genes. The cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aDNA sequence of interest, and a transcriptional and translationaltermination region functional in plants. The termination region may benative with the transcriptional initiation region, may be native withthe DNA sequence of interest, or may be derived from another source.Convenient termination regions are available from the Ti-plasmid ofAgrobacterium tumefaciens, such, as the octopine synthase and nopalinesynthase termination regions (see also, Guerineau 1991; Proudfoot 1991;Sanfacon 1991; Mogen 1990; Munroe 1990; Ballas 1989; Joshi 1987).

“Coding sequence” refers to a DNA or RNA sequence that codes for aspecific amino acid sequence and excludes the non-coding sequences. Itmay constitute an “uninterrupted coding sequence”, i.e., lacking anintron, such as in a cDNA or it may include one or more introns boundedby appropriate splice junctions. An “intron” is a sequence of RNA whichis contained in the primary transcript but which is removed throughcleavage and re-ligation of the RNA within the cell to create the maturemRNA that can be translated into a protein.

The terms “open reading frame” and “ORF” refer to the amino acidsequence encoded between translation initiation and termination codonsof a coding sequence. The terms “initiation codon” and “terminationcodon” refer to a unit of three adjacent nucleotides (‘codon’) in acoding sequence that specifies initiation and chain termination,respectively, of protein synthesis (mRNA translation).

A “functional RNA” refers to an antisense RNA, double-stranded-RNA,ribozyme, or other RNA that is not translated.

The term “RNA transcript” refers to the product resulting from RNApolymerase catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” (mRNA) refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a single- or a double-stranded DNA that iscomplementary to and derived from mRNA.

“Transcription regulating nucleotide sequence”, “regulatory sequences”,and “suitable regulatory sequences”, each refer to nucleotide sequencesinfluencing the transcription, RNA processing or stability, ortranslation of the associated (or functionally linked) nucleotidesequence to be transcribed. The transcription regulating nucleotidesequence may have various localizations with the respect to thenucleotide sequences to be transcribed. The transcription regulatingnucleotide sequence may be located upstream (5′ non-coding sequences),within, or downstream (3′ non-coding sequences) of the sequence to betranscribed (e.g., a coding sequence). The transcription regulatingsequences may be selected from the group comprising enhancers,promoters, translation leader sequences, introns, 5′-untranslatedsequences, 3′-untranslated sequences, and polyadenylation signalsequences. They include natural and synthetic sequences as well assequences, which may be a combination of synthetic and naturalsequences. As is noted above, the term “transcription regulatingsequence” is not limited to promoters. However, preferably atranscription regulating nucleotide sequence of the invention comprisesat least one promoter sequence (e.g., a sequence localized upstream ofthe transcription start of a gene capable to induce transcription of thedownstream sequences). In one preferred embodiment the transcriptionregulating nucleotide sequence of the invention comprises the promotersequence of the corresponding gene and—optionally and preferably—thenative 5′-untranslated region of said gene. Furthermore, the3′-untranslated region and/or the polyadenylation region of said genemay also be employed.

“5′ non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency(Turner 1995).

“3′ non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., 1989.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

“Signal peptide” refers to the amino terminal extension of apolypeptide, which is translated in conjunction with the polypeptideforming a precursor peptide and which is required for its entrance intothe secretory pathway. The term “signal sequence” refers to a nucleotidesequence that encodes the signal peptide. The term “transit peptide” asused herein refers part of an expressed polypeptide (preferably to theamino terminal extension of a polypeptide), which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its entrance into a cell organelle (such as the plastids(e.g., chloroplasts) or mitochondria). The term “transit sequence”refers to a nucleotide sequence that encodes the transit peptide.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter” includes a minimal promoter that isa short DNA sequence comprised of a TATA box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. “Promoter” alsorefers to a nucleotide sequence that includes a minimal promoter plusregulatory elements that is capable of controlling the expression of acoding sequence or functional RNA. This type of promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence, which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue specificity of a promoter. It is capable ofoperating in both orientations (normal or flipped), and is capable offunctioning even when moved either upstream or downstream from thepromoter. Both enhancers and other upstream promoter elements bindsequence-specific DNA-binding proteins that mediate their effects.Promoters may be derived in their entirety from a native gene, or becomposed of different elements, derived from different promoters foundin nature, or even be comprised of synthetic DNA segments. A promotermay also contain DNA sequences that are involved in the binding ofprotein factors, which control the effectiveness of transcriptioninitiation in response to physiological or developmental conditions.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.,further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

Promoter elements, particularly a TATA element, that are inactive orthat have greatly reduced promoter activity in the absence of upstreamactivation are referred to as “minimal or core promoters.” In thepresence of a suitable transcription factor, the minimal promoterfunctions to permit transcription. A “minimal or core promoter” thusconsists only of all basal elements needed for transcription initiation,e.g., a TATA box and/or an initiator.

“Constitutive expression” refers to expression using a constitutive orregulated promoter.

By “tissue-independent,” “tissue-general,” or “constitutive” is intendedexpression in the cells throughout a plant at most times and in mosttissues. As with other promoters classified as “constitutive” (e.g.,ubiquitin), some variation in absolute levels of expression can existamong different tissues or stages. However, constitutive promotersgenerally are expressed at high or moderate levels in most, andpreferably all, tissues and most, and preferably all, developmentalstages. “Conditional” and “regulated expression” refer to expressioncontrolled by a regulated promoter.

“Constitutive promoter” refers to a promoter that is able to express theopen reading frame (ORF) that it controls in all or nearly all of theplant tissues during all or nearly all developmental stages of theplant. Each of the transcription-activating elements do not exhibit anabsolute tissue-specificity, but mediate transcriptional activation inmost plant parts at a level of at least 1% of the level reached in thepart of the plant in which transcription is most active.

“Regulated promoter” refers to promoters that direct gene expression notconstitutively, but in a temporally- and/or spatially-regulated manner,and includes both tissue-specific and inducible promoters. It includesnatural and synthetic sequences as well as sequences which may be acombination of synthetic and natural sequences. Different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. New promoters of various types useful in plantcells are constantly being discovered, numerous examples may be found inthe compilation by Okamuro et al. (1989). Typical regulated promotersuseful in plants include but are not limited to safener-induciblepromoters, promoters derived from the tetracycline-inducible system,promoters derived from salicylate-inducible systems, promoters derivedfrom alcohol-inducible systems, promoters derived fromglucocorticoid-inducible system, promoters derived frompathogen-inducible systems, and promoters derived fromecdysone-inducible systems.

“Tissue-specific promoter” refers to regulated promoters that are notexpressed in all plant cells but only in one or more cell types inspecific organs (such as leaves or seeds), specific tissues (such asembryo or cotyledon), or specific cell types (such as leaf parenchyma orseed storage cells). These also include promoters that are temporallyregulated, such as in early or late embryogenesis, during fruit ripeningin developing seeds or fruit, in fully differentiated leaf, or at theonset of senescence.

The term “root” in the context of the inventions means the usuallyunderground organ of a plant that lacks buds or leaves or nodes, absorbswater and mineral salts and usually it anchors the plant to the ground.The plant root consists of many cell types such as epidermal, root cap,columella, cortex, pericycle, vascular and root hair formingtrichoblasts, organized into tissues or regions of the root, forexample, the root tip, root epidermis, meristematic zone, primary root,lateral root, root hair, and vascular tissue. Transcription regulatingsequences isolated as root-specific or root-preferred may regulateexpression in one or a few of these cell types. This cell-specificactivity can be useful for specific applications such as regulatingmeristematic activity in only meristematic cell zone or expression of anematicidal gene in only the cell type that are contacted by thenematode pest.

The term “tissue-specific transcription” in the context of thisinvention in relation to a certain tissue or a group of tissue (e.g.,root and kernel) means the transcription of a nucleic acid sequence by atranscription regulating element in a way that transcription of saidnucleic acid sequence in said tissue or group of tissues contribute tomore than 90%, preferably more than 95%, more preferably more than 99%of the entire quantity of the RNA transcribed from said nucleic acidsequence in the entire plant during any of its developmental stage.

“Tissue-preferential transcription” in the context of this invention inrelation to a certain tissue or a group of tissue (e.g., root andkernel) means the transcription of a nucleic acid sequence by atranscription regulating element in a way that transcription of saidnucleic acid sequence in said tissue or group of tissues contribute tomore than 50%, preferably more than 70%, more preferably more than 80%of the entire quantity of the RNA transcribed from said nucleic acidsequence in the entire plant during any of its developmental stage.

“Inducible promoter” refers to those regulated promoters that can beturned on in one or more cell types by an external stimulus, such as achemical, light, hormone, stress, or a pathogen.

“Operably-linked” refers to the association of nucleic acid sequences onsingle nucleic acid fragment so that the function of one is affected bythe other. For example, a regulatory DNA sequence is said to be“operably linked to” or “associated with” a DNA sequence that codes foran RNA or a polypeptide if the two sequences are situated such that theregulatory DNA sequence affects expression of the coding DNA sequence(i.e., that the coding sequence or functional RNA is under thetranscriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

“Expression” refers to the transcription and/or translation of anendogenous gene, ORF or portion thereof, or a transgene in plants. Forexample, in the case of antisense constructs, expression may refer tothe transcription of the antisense DNA only. In addition, expressionrefers to the transcription and stable accumulation of sense (mRNA) orfunctional RNA. Expression may also refer to the production of protein.

“Specific expression” is the expression of gene products, which islimited to one or a few plant tissues (spatial limitation) and/or to oneor a few plant developmental stages (temporal limitation). It isacknowledged that hardly a true specificity exists: promoters seem to bepreferably switch on in some tissues, while in other tissues there canbe no or only little activity. This phenomenon is known as leakyexpression. However, with specific expression in this invention is meantpreferable expression in one or a few plant tissues.

The “expression pattern” of a promoter (with or without enhancer) is thepattern of expression levels, which shows where in the plant and in whatdevelopmental stage transcription is initiated by said promoter.Expression patterns of a set of promoters are said to be complementarywhen the expression pattern of one promoter shows little overlap withthe expression pattern of the other promoter. The level of expression ofa promoter can be determined by measuring the ‘steady state’concentration of a standard transcribed reporter mRNA. This measurementis indirect since the concentration of the reporter mRNA is dependentnot only on its synthesis rate, but also on the rate with which the mRNAis degraded. Therefore, the steady state level is the product ofsynthesis rates and degradation rates.

The rate of degradation can however be considered to proceed at a fixedrate when the transcribed sequences are identical, and thus this valuecan serve as a measure of synthesis rates. When promoters are comparedin this way, techniques available to those skilled in the art arehybridization S1-RNAse analysis, Northern blots and competitive RT-PCR.This list of techniques in no way represents all available techniques,but rather describes commonly used procedures used to analyzetranscription activity and expression levels of mRNA.

The analysis of transcription start points in practically all promotershas revealed that there is usually no single base at which transcriptionstarts, but rather a more or less clustered set of initiation sites,each of which accounts for some start points of the mRNA. Since thisdistribution varies from promoter to promoter the sequences of thereporter mRNA in each of the populations would differ from each other.Since each mRNA species is more or less prone to degradation, no singledegradation rate can be expected for different reporter mRNAs. It hasbeen shown for various eukaryotic promoter sequences that the sequencesurrounding the initiation site (‘initiator’) plays an important role indetermining the level of RNA expression directed by that specificpromoter. This includes also part of the transcribed sequences. Thedirect fusion of promoter to reporter sequences would therefore lead tosuboptimal levels of transcription.

A commonly used procedure to analyze expression patterns and levels isthrough determination of the ‘steady state’ level of proteinaccumulation in a cell. Commonly used candidates for the reporter gene,known to those skilled in the art are beta-glucuronidase (GUS),chloramphenicol acetyl transferase (CAT) and proteins with fluorescentproperties, such as green fluorescent protein (GFP) from Aequoravictoria. In principle, however, many more proteins are suitable forthis purpose, provided the protein does not interfere with essentialplant functions. For quantification and determination of localization anumber of tools are suited. Detection systems can readily be created orare available which are based on, e.g., immunochemical, enzymatic,fluorescent detection and quantification. Protein levels can bedetermined in plant tissue extracts or in intact tissue using in situanalysis of protein expression.

Generally, individual transformed lines with one chimeric promoterreporter construct will vary in their levels of expression of thereporter gene. Also frequently observed is the phenomenon that suchtransformants do not express any detectable product (RNA or protein).The variability in expression is commonly ascribed to ‘positioneffects’, although the molecular mechanisms underlying this inactivityare usually not clear.

“Overexpression” refers to the level of expression in transgenic cellsor organisms that exceeds levels of expression in normal oruntransformed (non-transgenic) cells or organisms.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of protein from anendogenous gene or a transgene.

“Gene silencing” refers to homology-dependent suppression of viralgenes, transgenes, or endogenous nuclear genes. Gene silencing may betranscriptional, when the suppression is due to decreased transcriptionof the affected genes, or post-transcriptional, when the suppression isdue to increased turnover (degradation) of RNA species homologous to theaffected genes (English 1996). Gene silencing includes virus-inducedgene silencing (Ruiz et al. 1998).

The terms “heterologous DNA sequence,” “exogenous DNA segment” or“heterologous nucleic acid,” as used herein, each refer to a sequencethat originates from a source foreign to the particular host cell or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell but has been modified through, for example, theuse of DNA shuffling. The terms also include non-naturally occurringmultiple copies of a naturally occurring DNA sequence. Thus, the termsrefer to a DNA segment that is foreign or heterologous to the cell, orhomologous to the cell but in a position within the host cell nucleicacid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides. A “homologous”DNA sequence is a DNA sequence that is naturally associated with a hostcell into which it is introduced.

“Homologous to” in the context of nucleotide sequence identity refers tothe similarity between the nucleotide sequences of two nucleic acidmolecules or between the amino acid sequences of two protein molecules.Estimates of such homology are provided by either DNA-DNA or DNA-RNAhybridization under conditions of stringency as is well understood bythose skilled in the art (as described in Haines and Higgins (eds.),Nucleic Acid Hybridization, IRL Press, Oxford, U.K.), or by thecomparison of sequence similarity between two nucleic acids or proteins.

The term “substantially similar” refers to nucleotide and amino acidsequences that represent functional and/or structural equivalents of thetranscription regulating sequences from maize or rice specificallydisclosed herein.

In its broadest sense, the term “substantially similar” when used hereinwith respect to a nucleotide sequence means that the nucleotide sequenceis part of a gene which encodes a polypeptide having substantially thesame structure and function as a polypeptide encoded by a gene for thereference nucleotide sequence, e.g., the nucleotide sequence comprises apromoter from a gene that is the ortholog of the gene corresponding tothe reference nucleotide sequence, as well as promoter sequences thatare structurally related the promoter sequences particularly exemplifiedherein, i.e., the substantially similar promoter sequences hybridize tothe complement of the promoter sequences exemplified herein under highor very high stringency conditions. For example, altered nucleotidesequences, which simply reflect the degeneracy of the genetic code butnonetheless encode amino acid sequences that are identical to aparticular amino acid sequence, are substantially similar to theparticular sequences. The term “substantially similar” also includesnucleotide sequences wherein the sequence has been modified, forexample, to optimize expression in particular cells, as well asnucleotide sequences encoding a variant polypeptide having one or moreamino acid substitutions relative to the (unmodified) polypeptideencoded by the reference sequence, which substitution(s) does not alterthe activity of the variant polypeptide relative to the unmodifiedpolypeptide.

In its broadest sense, the term “substantially similar” when used hereinwith respect to polypeptide means that the polypeptide has substantiallythe same structure and function as the reference polypeptide. Inaddition, amino acid sequences that are substantially similar to aparticular sequence are those wherein overall amino acid identity is atleast 65% or greater to the instant sequences. Modifications that resultin equivalent nucleotide or amino acid sequences are well within theroutine skill in the art. The percentage of amino acid sequence identitybetween the substantially similar and the reference polypeptide is atleast 65%, 66%, 67%, 68%, 69%, 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, andeven 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, up to atleast 99%, wherein the reference polypeptide is a polypeptide encoded bya gene with a promoter having any one of SEQ ID NOs: 1, 2, 3, 6, 7, 8,11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58,61, 62, 63, 66, 67, 68, 71, 72, or 73, a nucleotide sequence comprisingan open reading frame having any one of SEQ ID NOs: 4, 9, 17, 25, 30,59, 64, 69, or 74, which encodes one of SEQ ID NOs: 5, 10, 18, 26, 31,60, 65, 70, or 75. One indication that two polypeptides aresubstantially similar to each other, besides having substantially thesame function, is that an agent, e.g., an antibody, which specificallybinds to one of the polypeptides, specifically binds to the other.

Sequence comparisons maybe carried out using a Smith-Waterman sequencealignment algorithm (see e.g., Waterman (1995) or http://wwwhto.usc.edu/software/seqaln/index.html). The localS program, version1.16, is preferably used with following parameters: match: 1, mismatchpenalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2.

Moreover, a nucleotide sequence that is “substantially similar” to areference nucleotide sequence is said to be “equivalent” to thereference nucleotide sequence. The skilled artisan recognizes thatequivalent nucleotide sequences encompassed by this invention can alsobe defined by their ability to hybridize, under low, moderate and/orstringent conditions (e.g., 0.1×SSC, 0.1% SDS, 65° C.), with thenucleotide sequences that are within the literal scope of the instantclaims.

What is meant by “substantially the same activity” when used inreference to a polynucleotide or polypeptide fragment is that thefragment has at least 65%, 66%, 67%, 68%, 69%, 70%, e.g., 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, up to at least 99% of the activity of the full lengthpolynucleotide or full length polypeptide.

“Target gene” refers to a gene on the replicon that expresses thedesired target coding sequence, functional RNA, or protein. The targetgene is not essential for replicon replication. Additionally, targetgenes may comprise native non-viral genes inserted into a non-nativeorganism, or chimeric genes, and will be under the control of suitableregulatory sequences. Thus, the regulatory sequences in the target genemay come from any source, including the virus. Target genes may includecoding sequences that are either heterologous or homologous to the genesof a particular plant to be transformed. However, target genes do notinclude native viral genes. Typical target genes include, but are notlimited to genes encoding a structural protein, a seed storage protein,a protein that conveys herbicide resistance, and a protein that conveysinsect resistance. Proteins encoded by target genes are known as“foreign proteins”. The expression of a target gene in a plant willtypically produce an altered plant trait.

The term “altered plant trait” means any phenotypic or genotypic changein a transgenic plant relative to the wild-type or non-transgenic planthost.

“Replication gene” refers to a gene encoding a viral replicationprotein. In addition to the ORF of the replication protein, thereplication gene may also contain other overlapping or non-overlappingORF(s), as are found in viral sequences in nature. While not essentialfor replication, these additional ORFs may enhance replication and/orviral DNA accumulation. Examples of such additional ORFs are AC3 and AL3in ACMV and TGMV geminiviruses, respectively.

“Chimeric trans-acting replication gene” refers either to a replicationgene in which the coding sequence of a replication protein is under thecontrol of a regulated plant promoter other than that in the nativeviral replication gene, or a modified native viral replication gene, forexample, in which a site specific sequence(s) is inserted in the 5′transcribed but untranslated region. Such chimeric genes also includeinsertion of the known sites of replication protein binding between thepromoter and the transcription start site that attenuate transcriptionof viral replication protein gene.

“Chromosomally-integrated” refers to the integration of a foreign geneor DNA construct into the host DNA by covalent bonds. Where genes arenot “chromosomally integrated” they may be “transiently expressed.”Transient expression of a gene refers to the expression of a gene thatis not integrated into the host chromosome but functions independently,either as part of an autonomously replicating plasmid or expressioncassette, for example, or as part of another biological system such as avirus.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. Host cells containing the transformed nucleic acidfragments are referred to as “transgenic” cells, and organismscomprising transgenic cells are referred to as “transgenic organisms”.Examples of methods of transformation of plants and plant cells includeAgrobacterium-mediated transformation (De Blaere 1987) and particlebombardment technology (U.S. Pat. No. 4,945,050). Whole plants may beregenerated from transgenic cells by methods well known to the skilledartisan (see, for example, Fromm 1990).

“Transformed,” “transgenic,” and “recombinant” refer to a host organismsuch as a bacterium or a plant into which a heterologous nucleic acidmolecule has been introduced. The nucleic acid molecule can be stablyintegrated into the genome generally known in the art and are disclosed(Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999. Knownmethods of PCR include, but are not limited to, methods using pairedprimers, nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially mismatchedprimers, and the like. For example, “transformed,” “transformant,” and“transgenic” plants or calli have been through the transformationprocess and contain a foreign gene integrated into their chromosome. Theterm “untransformed” refers to normal plants that have not been throughthe transformation process.

“Transiently transformed” refers to cells in which transgenes andforeign DNA have been introduced (for example, by such methods asAgrobacterium-mediated transformation or biolistic bombardment), but notselected for stable maintenance.

“Stably transformed” refers to cells that have been selected andregenerated on a selection media following transformation.

“Transient expression” refers to expression in cells in which a virus ora transgene is introduced by viral infection or by such methods asAgrobacterium-mediated transformation, electroporation, or biolisticbombardment, but not selected for its stable maintenance.

“Genetically stable” and “heritable” refer to chromosomally-integratedgenetic elements that are stably maintained in the plant and stablyinherited by progeny through successive generations.

“Primary transformant” and “T0 generation” refer to transgenic plantsthat are of the same genetic generation as the tissue which wasinitially transformed (i.e., not having gone through meiosis andfertilization since transformation).

“Secondary transformants” and the “T1, T2, T3, etc. generations” referto transgenic plants derived from primary transformants through one ormore meiotic and fertilization cycles. They may be derived byself-fertilization of primary or secondary transformants or crosses ofprimary or secondary transformants with other transformed oruntransformed plants.

“Wild-type” refers to a virus or organism found in nature without anyknown mutation.

“Genome” refers to the complete genetic material of an organism.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base, which is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides, which have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer 1991; Ohtsuka 1985; Rossolini 1994). A“nucleic acid fragment” is a fraction of a given nucleic acid molecule.In higher plants, deoxyribonucleic acid (DNA) is the genetic materialwhile ribonucleic acid (RNA) is involved in the transfer of informationcontained within DNA into proteins. The term “nucleotide sequence”refers to a polymer of DNA or RNA which can be single- ordouble-stranded, optionally containing synthetic, non-natural or alterednucleotide bases capable of incorporation into DNA or RNA polymers. Theterms “nucleic acid” or “nucleic acid sequence” may also be usedinterchangeably with gene, cDNA, DNA and RNA encoded by a gene.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. In the context of the present invention,an “isolated” or “purified” DNA molecule or an “isolated” or “purified”polypeptide is a DNA molecule or polypeptide that, by the hand of man,exists apart from its native environment and is therefore not a productof nature. An isolated DNA molecule or polypeptide may exist in apurified form or may exist in a non-native environment such as, forexample, a transgenic host cell. For example, an “isolated” or“purified” nucleic acid molecule or protein, or biologically activeportion thereof, is substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. Preferably, an “isolated” nucleic acid is free of sequences(preferably protein encoding sequences) that naturally flank the nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived. A protein that is substantially free of cellular materialincludes preparations of protein or polypeptide having less than about30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When theprotein of the invention, or biologically active portion thereof, isrecombinantly produced, preferably culture medium represents less thanabout 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors ornon-protein of interest chemicals.

The nucleotide sequences of the invention include both the naturallyoccurring sequences as well as mutant (variant) forms. Such variantswill continue to possess the desired activity, i.e., either promoteractivity or the activity of the product encoded by the open readingframe of the non-variant nucleotide sequence.

The term “variant” with respect to a sequence (e.g., a polypeptide ornucleic acid sequence such as—for example—a transcription regulatingnucleotide sequence of the invention) is intended to mean substantiallysimilar sequences. For nucleotide sequences comprising an open readingframe, variants include those sequences that, because of the degeneracyof the genetic code, encode the identical amino acid sequence of thenative protein. Naturally occurring allelic variants such as these canbe identified with the use of well-known molecular biology techniques,as, for example, with polymerase chain reaction (PCR) and hybridizationtechniques. Variant nucleotide sequences also include syntheticallyderived nucleotide sequences, such as those generated, for example, byusing site-directed mutagenesis and for open reading frames, encode thenative protein, as well as those that encode a polypeptide having aminoacid substitutions relative to the native protein. Generally, nucleotidesequence variants of the invention will have at least 40, 50, 60, to70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99%nucleotide sequence identity to the native (wild type or endogenous)nucleotide sequence.

“Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acid sequences that encode identical oressentially identical amino acid sequences, or where the nucleic acidsequence does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenpolypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGGall encode the amino acid arginine. Thus, at every position where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded protein.Such nucleic acid variations are “silent variations” which are onespecies of “conservatively modified variations.” Every nucleic acidsequence described herein, which encodes a polypeptide, also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except ATG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid, which encodes apolypeptide, is implicit in each described sequence.

The nucleic acid molecules of the invention can be “optimized” forenhanced expression in plants of interest (see, for example, WO91/16432; Perlak 1991; Murray 1989). In this manner, the open readingframes in genes or gene fragments can be synthesized utilizingplant-preferred codons (see, for example, Campbell & Gowri, 1990 for adiscussion of host-preferred codon usage). Thus, the nucleotidesequences can be optimized for expression in any plant. It is recognizedthat all or any part of the gene sequence may be optimized or synthetic.That is, synthetic or partially optimized sequences may also be used.Variant nucleotide sequences and proteins also encompass, sequences andprotein derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different codingsequences can be manipulated to create a new polypeptide possessing thedesired properties. In this manner, libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. Strategies for such DNA shuffling are known in the art (see, forexample, Stemmer 1994; Stemmer 1994; Crameri 1997; Moore 1997; Zhang1997; Crameri 1998; and U.S. Pat. Nos. 5,605,793 and 5,837,458).

By “variant” polypeptide is intended a polypeptide derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Such variants may resultfrom, for example, genetic polymorphism or from human manipulation.Methods for such manipulations are generally known in the art.

Thus, the polypeptides may be altered in various ways including aminoacid substitutions, deletions, truncations, and insertions. Methods forsuch manipulations are generally known in the art. For example, aminoacid sequence variants of the polypeptides can be prepared by mutationsin the DNA. Methods for mutagenesis and nucleotide sequence alterationsare well known in the art (see, for example, Kunkel 1985; Kunkel 1987;U.S. Pat. No. 4,873,192; Walker & Gaastra, 1983 and the references citedtherein). Guidance as to appropriate amino acid substitutions that donot affect biological activity of the protein of interest may be foundin the model of Dayhoff et al. (1978). Conservative substitutions, suchas exchanging one amino acid with another having similar properties, arepreferred.

Individual substitutions deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are “conservatively modified variations,” where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following five groupseach contain amino acids that are conservative substitutions for oneanother: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L),Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan(W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine(R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid(E), Asparagine (N), Glutamine (Q). See also, Creighton, 1984. Inaddition, individual substitutions, deletions or additions which alter,add or delete a single amino acid or a small percentage of amino acidsin an encoded sequence are also “conservatively modified variations.”

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to anucleotide sequence of interest, which is—optionally operably linked totermination signals and/or other regulatory elements. An expressioncassette may also comprise sequences required for proper translation ofthe nucleotide sequence. The coding region usually codes for a proteinof interest but may also code for a functional RNA of interest, forexample antisense RNA or a nontranslated RNA, in the sense or antisensedirection. The expression cassette comprising the nucleotide sequence ofinterest may be chimeric, meaning that at least one of its components isheterologous with respect to at least one of its other components. Theexpression cassette may also be one, which is naturally occurring buthas been obtained in a recombinant form useful for heterologousexpression. An expression cassette may be assembled entirelyextracellularly (e.g., by recombinant cloning techniques). However, anexpression cassette may also be assembled using in part endogenouscomponents. For example, an expression cassette may be obtained byplacing (or inserting) a promoter sequence upstream of an endogenoussequence, which thereby becomes functionally linked and controlled bysaid promoter sequences. Likewise, a nucleic acid sequence to beexpressed may be placed (or inserted) downstream of an endogenouspromoter sequence thereby forming an expression cassette. The expressionof the nucleotide sequence in the expression cassette may be under thecontrol of a constitutive promoter or of an inducible promoter whichinitiates transcription only when the host cell is exposed to someparticular external stimulus. In the case of a multicellular organism,the promoter can also be specific to a particular tissue or organ orstage of development (e.g., root/kernel specific or preferential).

“Vector” is defined to include, inter alia, any plasmid, cosmid, phageor Agrobacterium binary vector in double or single stranded linear orcircular form which may or may not be self transmissible or mobilizable,and which can transform prokaryotic or eukaryotic host either byintegration into the cellular genome or exist extrachromosomally (e.g.autonomous replicating plasmid with an origin of replication).

Specifically included are shuttle vectors by which is meant a DNAvehicle capable, naturally or by design, of replication in two differenthost organisms, which may be selected from Actinomycetes and relatedspecies, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast orfungal cells).

Preferably the nucleic acid in the vector is under the control of, andoperably linked to, an appropriate promoter or other regulatory elementsfor transcription in a host cell such as a microbial, e.g. bacterial, orplant cell. The vector may be a bi-functional expression vector whichfunctions in multiple hosts. In the case of genomic DNA, this maycontain its own promoter or other regulatory elements and in the case ofcDNA this may be under the control of an appropriate promoter or otherregulatory elements for expression in the host cell.

“Cloning vectors” typically contain one or a small number of restrictionendonuclease recognition sites at which foreign DNA sequences can beinserted in a determinable fashion without loss of essential biologicalfunction of the vector, as well as a marker gene that is suitable foruse in the identification and selection of cells transformed with thecloning vector. Marker genes typically include genes that providetetracycline resistance, hygromycin resistance or ampicillin resistance.

A “transgenic plant” is a plant having one or more plant cells thatcontain an expression vector.

“Plant tissue” includes differentiated and undifferentiated tissues orplants, including but not limited to roots, stems, shoots, leaves,pollen, seeds, tumor tissue and various forms of cells and culture suchas single cells, protoplast, embryos, and callus tissue. The planttissue may be in plants or in organ, tissue or cell culture.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

-   (a) As used herein, “reference sequence” is a defined sequence used    as a basis for sequence comparison. A reference sequence may be a    subset or the entirety of a specified sequence; for example, as a    segment of a full-length cDNA or gene sequence, or the complete cDNA    or gene sequence.-   (b) As used herein, “comparison window” makes reference to a    contiguous and specified segment of a polynucleotide sequence,    wherein the polynucleotide sequence in the comparison window may    comprise additions or deletions (i.e., gaps) compared to the    reference sequence (which does not comprise additions or deletions)    for optimal alignment of the two sequences. Generally, the    comparison window is at least 20 contiguous nucleotides in length,    and optionally can be 30, 40, 50, 100, or longer. Those of skill in    the art understand that to avoid a high similarity to a reference    sequence due to inclusion of gaps in the polynucleotide sequence a    gap penalty is typically introduced and is subtracted from the    number of matches. Methods of alignment of sequences for comparison    are well known in the art. Thus, the determination of percent    identity between any two sequences can be accomplished using a    mathematical algorithm. Preferred, non-limiting examples of such    mathematical algorithms are the algorithm of Myers and Miller, 1988;    the local homology algorithm of Smith et al. 1981; the homology    alignment algorithm of Needleman and Wunsch 1970; the    search-for-similarity-method of Pearson and Lipman 1988; the    algorithm of Karlin and Altschul, 1990, modified as in Karlin and    Altschul, 1993.    -   Computer implementations of these mathematical algorithms can be        utilized for comparison of sequences to determine sequence        identity. Such implementations include, but are not limited to:        CLUSTAL in the PC/Gene program (available from Intelligenetics,        Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,        BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics        Software Package, Version 8 (available from Genetics Computer        Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments        using these programs can be performed using the default        parameters. The CLUSTAL program is well described (Higgins 1988,        1989; Corpet 1988; Huang 1992; Pearson 1994). The ALIGN program        is based on the algorithm of Myers and Miller, supra. The BLAST        programs of Altschul et al., 1990, are based on the algorithm of        Karlin and Altschul supra.    -   Software for performing BLAST analyses is publicly available        through the National Center for Biotechnology Information        (http://www.ncbi.nlm.nih.gov/). This algorithm involves first        identifying high scoring sequence pairs (HSPs) by identifying        short words of length W in the query sequence, which either        match or satisfy some positive-valued threshold score T when        aligned with a word of the same length in a database sequence. T        is referred to as the neighborhood word score threshold        (Altschul 1990). These initial neighborhood word hits act as        seeds for initiating searches to find longer HSPs containing        them. The word hits are then extended in both directions along        each sequence for as far as the cumulative alignment score can        be increased. Cumulative scores are calculated using, for        nucleotide sequences, the parameters M (reward score for a pair        of matching residues; always >0) and N (penalty score for        mismatching residues; always <0). For amino acid sequences, a        scoring matrix is used to calculate the cumulative score.        Extension of the word hits in each direction are halted when the        cumulative alignment score falls off by the quantity X from its        maximum achieved value, the cumulative score goes to zero or        below due to the accumulation of one or more negative-scoring        residue alignments, or the end of either sequence is reached.    -   In addition to calculating percent sequence identity, the BLAST        algorithm also performs a statistical analysis of the similarity        between two sequences (see, e.g., Karlin & Altschul (1993). One        measure of similarity provided by the BLAST algorithm is the        smallest sum probability (P(N)), which provides an indication of        the probability by which a match between two nucleotide or amino        acid sequences would occur by chance. For example, a test        nucleic acid sequence is considered similar to a reference        sequence if the smallest sum probability in a comparison of the        test nucleic acid sequence to the reference nucleic acid        sequence is less than about 0.1, more preferably less than about        0.01, and most preferably less than about 0.001.    -   To obtain gapped alignments for comparison purposes, Gapped        BLAST (in BLAST 2.0) can be utilized as described in Altschul et        al. 1997. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to        perform an iterated search that detects distant relationships        between molecules. See Altschul et al., supra. When utilizing        BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the        respective programs (e.g. BLASTN for nucleotide sequences,        BLASTX for proteins) can be used. The BLASTN program (for        nucleotide sequences) uses as defaults a wordlength (W) of 11,        an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a        comparison of both strands. For amino acid sequences, the BLASTP        program uses as defaults a wordlength (W) of 3, an        expectation (E) of 10, and the BLOSUM62 scoring matrix (see        Henikoff & Henikoff, 1989). See http://www.ncbi.nlm.nih.gov.        Alignment may also be performed manually by inspection.    -   For purposes of the present invention, comparison of nucleotide        sequences for determination of percent sequence identity to the        promoter sequences disclosed herein is preferably made using the        BlastN program (version 1.4.7 or later) with its default        parameters or any equivalent program. By “equivalent program” is        intended any sequence comparison program that, for any two        sequences in question, generates an alignment having identical        nucleotide or amino acid residue matches and an identical        percent sequence identity when compared to the corresponding        alignment generated by the preferred program.-   (c) As used herein, “sequence identity” or “identity” in the context    of two nucleic acid or polypeptide sequences makes reference to the    residues in the two sequences that are the same when aligned for    maximum correspondence over a specified comparison window. When    percentage of sequence identity is used in reference to proteins it    is recognized that residue positions which are not identical often    differ by conservative amino acid substitutions, where amino acid    residues are substituted for other amino acid residues with similar    chemical properties (e.g., charge or hydrophobicity) and therefore    do not change the functional properties of the molecule. When    sequences differ in conservative substitutions, the percent sequence    identity may be adjusted upwards to correct for the conservative    nature of the substitution. Sequences that differ by such    conservative substitutions are said to have “sequence similarity” or    “similarity.” Means for making this adjustment are well known to    those of skill in the art. Typically this involves scoring a    conservative substitution as a partial rather than a full mismatch,    thereby increasing the percentage sequence identity. Thus, for    example, where an identical amino acid is given a score of 1 and a    non-conservative substitution is given a score of zero, a    conservative substitution is given a score between zero and 1. The    scoring of conservative substitutions is calculated, e.g., as    implemented in the program PC/GENE (Intelligenetics, Mountain View,    Calif.).-   (d) As used herein, “percentage of sequence identity” means the    value determined by comparing two optimally aligned sequences over a    comparison window, wherein the portion of the polynucleotide    sequence in the comparison window may comprise additions or    deletions (i.e., gaps) as compared to the reference sequence (which    does not comprise additions or deletions) for optimal alignment of    the two sequences. The percentage is calculated by determining the    number of positions at which the identical nucleic acid base or    amino acid residue occurs in both sequences to yield the number of    matched positions, dividing the number of matched positions by the    total number of positions in the window of comparison, and    multiplying the result by 100 to yield the percentage of sequence    identity.-   (e) (i) The term “substantial identity” of polynucleotide sequences    means that a polynucleotide comprises a sequence that has at least    70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at    least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more    preferably at least 90%, 91%, 92%, 93%, or 94%, and most preferably    at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a    reference sequence using one of the alignment programs described    using standard parameters. One of skill in the art will recognize    that these values can be appropriately adjusted to determine    corresponding identity of proteins encoded by two nucleotide    sequences by taking into account codon degeneracy, amino acid    similarity, reading frame positioning, and the like. Substantial    identity of amino acid sequences for these purposes normally means    sequence identity of at least 70%, more preferably at least 80%,    90%, and most preferably at least 95%.    -   Another indication that nucleotide sequences are substantially        identical is if two molecules hybridize to each other under        stringent conditions (see below). Generally, stringent        conditions are selected to be about 5° C. lower than the thermal        melting point (T_(m)) for the specific sequence at a defined        ionic strength and pH. However, stringent conditions encompass        temperatures in the range of about 1° C. to about 20° C.,        depending upon the desired degree of stringency as otherwise        qualified herein. Nucleic acids that do not hybridize to each        other under stringent conditions are still substantially        identical if the polypeptides they encode are substantially        identical. This may occur, e.g., when a copy of a nucleic acid        is created using the maximum codon degeneracy permitted by the        genetic code. One indication that two nucleic acid sequences are        substantially identical is when the polypeptide encoded by the        first nucleic acid is immunologically cross reactive with the        polypeptide encoded by the second nucleic acid.    -   (ii) The term “substantial identity” in the context of a peptide        indicates that a peptide comprises a sequence with at least 70%,        71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%,        81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably        at least 90%, 91%, 92%, 93%, or 94%, or even more preferably,        95%, 96%, 97%, 98% or 99%, sequence identity to the reference        sequence over a specified comparison window. Preferably, optimal        alignment is conducted using the homology alignment algorithm of        Needleman and Wunsch (1970). An indication that two peptide        sequences are substantially identical is that one peptide is        immunologically reactive with antibodies raised against the        second peptide. Thus, a peptide is substantially identical to a        second peptide, for example, where the two peptides differ only        by a conservative substitution.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

As noted above, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridization are sequence dependent, andare different under different environmental parameters. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA-DNA hybrids, the T_(m) can be approximated from theequation of Meinkoth and Wahl, 1984:

T _(m)=81.5° C.+16.6(log₁₀ M)+0.41(% GC)-0.61(% form)−500/L

where M is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % form is the percentageof formamide in the hybridization solution, and L is the length of thehybrid in base pairs. T_(m) is reduced by about 1° C. for each 1% ofmismatching; thus, T_(m), hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with >90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the thermal melting point I for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point I; moderately stringentconditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C. lower than the thermal melting point I; low stringency conditions canutilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C.lower than the thermal melting point I. Using the equation,hybridization and wash compositions, and desired T, those of ordinaryskill will understand that variations in the stringency of hybridizationand/or wash solutions are inherently described. If the desired degree ofmismatching results in a T of less than 45° C. (aqueous solution) or 32°C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen, 1993.Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point T_(m)for the specific sequence at a defined ionic strength and pH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4 to 6×SSC at 40° C. for15 minutes. For short probes (e.g., about 10 to 50 nucleotides),stringent conditions typically involve salt concentrations of less thanabout 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration(or other salts) at pH 7.0 to 8.3, and the temperature is typically atleast about 30° C. and at least about 60° C. for long robes (e.g., >50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. In general, a signalto noise ratio of 2× (or higher) than that observed for an unrelatedprobe in the particular hybridization assay indicates detection of aspecific hybridization. Nucleic acids that do not hybridize to eachother under stringent conditions are still substantially identical ifthe proteins that they encode are substantially identical. This occurs,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1 MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone orthologous nucleotide sequences that aresubstantially identical to reference nucleotide sequences of the presentinvention: a reference nucleotide sequence preferably hybridizes to thereference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C.,more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirablystill in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C.

“DNA shuffling” is a method to introduce mutations or rearrangements,preferably randomly, in a DNA molecule or to generate exchanges of DNAsequences between two or more DNA molecules, preferably randomly. TheDNA molecule resulting from DNA shuffling is a shuffled DNA moleculethat is a non-naturally occurring DNA molecule derived from at least onetemplate DNA molecule. The shuffled DNA preferably encodes a variantpolypeptide modified with respect to the polypeptide encoded by thetemplate DNA, and may have an altered biological activity with respectto the polypeptide encoded by the template DNA.

“Recombinant DNA molecule’ is a combination of DNA sequences that arejoined together using recombinant DNA technology and procedures used tojoin together DNA sequences as described, for example, in Sambrook etal., 1989.

The word “plant” refers to any plant, particularly to agronomicallyuseful plants (e.g., seed plants), and “plant cell” is a structural andphysiological unit of the plant, which comprises a cell wall but mayalso refer to a protoplast. The plant cell may be in form of an isolatedsingle cell or a cultured cell, or as a part of higher organized unitsuch as, for example, a plant tissue, or a plant organ differentiatedinto a structure that is present at any stage of a plant's development.Such structures include one or more plant organs including, but are notlimited to, fruit, shoot, stem, leaf, flower petal, etc. Preferably, theterm “plant” includes whole plants, shoot vegetative organs/structures(e.g. leaves, stems and tubers), roots, flowers and floralorgans/structures (e.g. bracts, sepals, petals, stamens, carpels,anthers and ovules), seeds (including embryo, endosperm, and seed coat)and fruits (the mature ovary), plant tissues (e.g. vascular tissue,ground tissue, and the like) and cells (e.g. guard cells, egg cells,trichomes and the like), and progeny of same.

The class of plants that can be used in the method of the invention isgenerally as broad as the class of higher and lower plants amenable totransformation techniques, including angiosperms (monocotyledonous anddicotyledonous plants), gymnosperms, ferns, and multicellular algae. Itincludes plants of a variety of ploidy levels, including aneuploid,polyploid, diploid, haploid and hemizygous. Included within the scope ofthe invention are all genera and species of higher and lower plants ofthe plant kingdom. Included are furthermore the mature plants, seed,shoots and seedlings, and parts, propagation material (for example seedsand fruit) and cultures, for example cell cultures, derived therefrom.

Annual, perennial, monocotyledonous and dicotyledonous plants arepreferred host organisms for the generation of transgenic plants. Theuse of the recombination system, or method according to the invention isfurthermore advantageous in all ornamental plants, forestry, fruit, orornamental trees, flowers, cut flowers, shrubs or turf. Said plant mayinclude—but shall not be limited to—bryophytes such as, for example,Hepaticae (hepaticas) and Musci (mosses); pteridophytes such as ferns,horsetail and clubmosses; gymnosperms such as conifers, cycads, ginkgoand Gnetaeae; algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae,Myxophyceae, Xanthophyceae, Bacillariophyceae (diatoms) andEuglenophyceae.

Plants for the purposes of the invention may comprise the families ofthe Rosaceae such as rose, Ericaceae such as rhododendrons and azaleas,Euphorbiaceae such as poinsettias and croton, Caryophyllaceae such aspinks, Solanaceae such as petunias, Gesneriaceae such as African violet,Balsaminaceae such as touch-me-not, Orchidaceae such as orchids,Iridaceae such as gladioli, iris, freesia and crocus, Compositae such asmarigold, Geraniaceae such as geraniums, Liliaceae such as Drachaena,Moraceae such as ficus, Araceae such as philodendron and many others.

The transgenic plants according to the invention are furthermoreselected from among dicotyledonous crop plants such as, for example,from the families of the Leguminosae such as pea, alfalfa and soybean;the family of the Umbelliferae, particularly the genus Daucus (veryparticularly the species carota (carrot)) and Apium (very particularlythe species graveolens var. dulce (celery)) and many others; the familyof the Solanaceae, particularly the genus Lycopersicon, veryparticularly the species esculentum (tomato) and the genus Solanum, veryparticularly the species tuberosum (potato) and melongena (aubergine),tobacco and many others; and the genus Capsicum, very particularly thespecies annum (pepper) and many others; the family of the Leguminosae,particularly the genus Glycine, very particularly the species max(soybean) and many others; and the family of the Cruciferae,particularly the genus Brassica, very particularly the species napus(oilseed rape), campestris (beet), oleracea cv Tastie (cabbage),oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli);and the genus Arabidopsis, very particularly the species thaliana andmany others; the family of the Compositae, particularly the genusLactuca, very particularly the species sativa (lettuce) and many others.Further preferred are trees such as apple, pear, quince, plum, cherry,peach, nectarine, apricot, papaya, mango, and other woody speciesincluding coniferous and deciduous trees such as poplar, pine, sequoia,cedar, oak, etc.

Most preferably, the transgenic plants according to the invention may beselected among monocotyledonous crop plants. The term “monocotyledonousplant” when referring to a transgenic plant according to the inventionor to the source of the transcription regulating sequences of theinvention is intended to comprise all genera, families and species ofmonocotyledonous plants. Preferred are Gramineae plants such as, forexample, cereals such as maize, rice, wheat, barley, sorghum, millet,rye, triticale, or oats, and other non-cereal monocotyledonous plantssuch as sugarcane or banana. Especially preferred are corn (maize),rice, barley, wheat, rye, and oats. Most preferred are all varieties ofthe specie Zea mays and Oryza sativa.

“Significant increase” is an increase that is larger than the margin oferror inherent in the measurement technique, preferably an increase byabout 2-fold or greater.

“Significantly less” means that the decrease is larger than the marginof error inherent in the measurement technique, preferably a decrease byabout 2-fold or greater.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for isolated nucleic acid moleculescomprising a plant nucleotide sequence that directs transcription of anoperably linked nucleic acid fragment in a plant cell, preferably inmonocotyledonous plants. Specifically, the present invention providesexpression cassettes for regulating expression in monocotyledonousplants comprising

-   i) at least one transcription regulating nucleotide sequence of a    monocotyledonous plant gene, said monocotyledonous plant gene    selected from the group of genes consisting of    caffeoyl-CoA-O-methyltransferase genes, C8,7-sterol isomerase genes,    hydroxyproline-rich glycoprotein (HRGP) genes, lactate dehydrogenase    genes, and chloroplast protein like 12 genes, and functionally    linked thereto-   ii) at least one nucleic acid sequence which is heterologous in    relation to said transcription regulating sequence.

Preferably a transcription regulating nucleotide sequence of theinvention comprises at least one promoter sequence of the respectivegene (e.g., a sequence localized upstream of the transcription start ofthe respective gene capable to induce transcription of the downstreamsequences). The transcription regulating nucleotide sequence of theinvention may comprise the promoter sequence of said genes but mayfurther comprise other elements such as the 5′-untranslated sequence,enhancer, introns etc. Preferably, said promoter sequence directstranscription of an operably linked nucleic acid segment in a plant orplant cell e.g., a linked plant DNA comprising an open reading frame fora structural or regulatory gene.

The transcription regulating sequences of the inventions can be combinedwith various 5′-untranslated regions, intron (preferably expressionenhancing introns), and transcription terminations sequences (asdescribed below in more detail). It has been shown that the tissuespecificity of the transcription regulating sequences of the inventioncan be advantageously modulated by the combination with introns and/ortranscription termination sequences. In most combinations the resultingexpression cassettes exhibit a preferential or specific expression inroot and kernel. However other expression specificities (e.g.,constitutive expression) can be achieved. The transcription regulatingsequences with expression activity in roots may be useful for alterationof the function of root tissue, modification of growth rate, improvementof resistance to root preferred pathogens, pests, herbicides or adverseweather conditions, for detoxification of soil as well as for broadeningthe range of soils or environments in which said plant may grow. Rootabundant or root specific gene expression would provide a mechanismaccording to which morphology and metabolism may be altered to improvethe yield and to produce useful proteins in greater amounts.

However, in some combinations, the transcriptions regulating sequencemay exhibit a strong constitutive expression profile. Constitutivepromoters are favored in situations where expression in all (or most)tissues during all (or most) times of the plant development is required.Other tissue specificities may be possible depending on the regulatoryelements used in combination with the transcription regulating sequencesof the invention.

The following Table 1 illustrates the genes from which the promoters ofthe invention are preferably isolated, the function of said genes, thecDNA encoded by said genes, and the protein (ORF) encoded by said genes.

TABLE 1 Genes from which the promoters of the invention are preferablyisolated, putative function of said genes, cDNA and the protein encodedby said genes. Preferred Promotor mRNA locus ID Proteine ID Gene ProductSpecie SEQ ID cDNA SEQ ID Protein SEQ ID Caffeoyl-CoA-O- Oryza sativaSEQ ID NO: 1, 2, 3 AB023482.2 BAA78733 methyltransferase SEQ ID NO: 4SEQ ID NO: 5 Caffeoyl-CoA-O- Zea mays SEQ ID NO: 66, 67, 68 SEQ ID NO:69 SEQ ID NO: 70 methyltransferase C-8,7-sterol- Oryza sativa SEQ ID NO:6, 7, 8 NM_183458 NP_908347 isomerase SEQ ID NO: 9 SEQ ID NO: 10Hydroxyproline- Zea mays SEQ ID NO: S45164 AAB23539 rich glycoprotein11, 12, 13, 14, 15, 16 SEQ ID NO: 17 SEQ ID NO: 18 Hydroxyproline- ZeaSEQ ID NO: 71, 72, 73 SEQ ID NO: 74 SEQ ID NO: 75 rich glycoproteindiploperennis Lactate- Zea mays SEQ ID NO: Z11754 CAA77808 dehydrogenase19, 20, 21, 22, 23, 24 SEQ ID NO: 25 SEQ ID NO: 26 Lactate- Oryza sativaSEQ ID NO: 56, 57, Os06g01590 Os06g01590 dehydrogenase 58, 61, 62, 63SEQ ID NO: 59 SEQ ID NO: 60 Os02g01510 Os02g01510 SEQ ID NO: 64 SEQ IDNO: 65 Chloroplast protein Oryza sativa SEQ ID NO: 27, 28, 29 AP002881BAB19776 12 lie protein SEQ ID NO: 30 SEQ ID NO: 31

Preferably, the transcription regulating nucleotide sequence isobtainable from monocotyledonous plant genomic DNA from a gene encodinga polypeptide which

-   a1) comprises at least one (preferably at least 2 or 3, more    preferably at least 4 or 5, most preferably all) sequence motif of a    monocotyledonous plant lactate dehydrogenase protein selected from    the group consisting of the amino acid sequences

i) (SEQ ID NO: 76) SLSELGFDA, ii) (SEQ ID NO: 77) VIGAGNVGMA, iii)(SEQ ID NO: 78) IVTAGARQI, iv) (SEQ ID NO: 79) L(F/Y)RKIVP, v)(SEQ ID NO: 80) GFPASRV, vi) (SEQ ID NO: 81) RF(L/I)AEHL, vii)(SEQ ID NO: 82) QAYMVGEH, viii) (SEQ ID NO: 83) ALEGIRRAV,  and ix)(SEQ ID NO: 84) GYSVAS(L/I)A,

-   -   or

-   b1) is encoding a lactate dehydrogenase protein from a    monocotyledonous plant having an amino acid sequence identity of at    least 90%, preferably at least 95%, more preferably at least 98% to    a polypeptide selected from the group described by SEQ ID NO: 26, 60    and 65, or

-   a2) comprises at least one (preferably at least 2 or 3, more    preferably at least 4 or 5, most preferably all) sequence motif of a    monocotyledonous plant caffeoyl-CaA-O-methyltransferase protein    selected from the group consisting of the amino acid sequences

x) (SEQ ID NO: 85) EQKTRHSE, xi) (SEQ ID NO: 86) L(I/L)KLIGAK, xii)(SEQ ID NO: 87) KTMEIGVY, xiii)  (SEQ ID NO: 88) HERL(L/M)KLV, xiv)(SEQ ID NO: 89) CQLPVGDG,  and xv) (SEQ ID NO: 90) TLCRRVK,

-   -   or

-   b2) is encoding a caffeoyl-CaA-O-methyltransferase protein from a    monocotyledonous plant having an amino acid sequence identity of at    least 90%, preferably at least 95%, more preferably at least 98% to    a polypeptide selected from the group described by SEQ ID NOs: 5 and    70, or

-   b3) is encoding a hydroxyproline-rich glycoprotein from a    monocotyledonous plant having an amino acid sequence identity of at    least 90%, preferably at least 95%, more preferably at least 98% to    a polypeptide selected from the group described by SEQ ID NOs: 18    and 75, or

-   b4) is encoding a C-8,7-stereol-isomerase protein from a    monocotyledonous plant having an amino acid sequence identity of at    least 90%, preferably at least 95%, more preferably at least 98% to    a polypeptide selected described by SEQ ID NO: 10, or

-   b5) is encoding a Chloroplast protein 12 like protein from a    monocotyledonous plant having an amino acid sequence identity of at    least 90%, preferably at least 95%, more preferably at least 98% to    a polypeptide described by SEQ ID NO: 31.

Preferably functional equivalent of the transcription regulatingnucleotide sequence can be obtained or is obtainable from plant genomicDNA from a gene expressing a mRNA described by a cDNA which issubstantially similar and preferably has at least 70%, preferably 80%,more preferably 90%, most preferably 95% sequence identity to a sequencedescribed by any one of SEQ ID NOs: 4, 9, 17, 25, 30, 59, 64, 69, or 74,respectively, or a fragment of said transcription regulating nucleotidesequence which exhibits the same promoter activity (e.g.,root/kernel-preferential or root/kernel-specific or constitutiveexpression activity).

Preferably, the transcription regulating nucleotide sequence is from acorn (Zea mays) or rice (Oryza sativa) plant. Even more preferably thetranscription regulating nucleotide sequence is from a plant geneselected from the group of genes consisting of Oryza sativacaffeoyl-CoA-O-methyltransferase genes, Oryza sativa C8,7-sterolisomerase genes, Zea may hydroxyproline-rich glycoprotein (HRGP) genes,Zea mays lactate dehydrogenase genes, Oryza sativa chloroplast protein12 like genes and functional equivalents thereof. The functionalequivalent gene is preferably encoding a polypeptide which has at least90% amino acid sequence identity, preferably at least 95% amino acidsequence identity, more preferably at least 98% amino acid sequenceidentity to a polypeptide selected from the group described by SEQ IDNOs: 5, 10, 18, 26, 31, 60, 65, 70, and 75.

Some of the transcription regulating sequences of the invention providedherein are novel as such (i.e. as isolated nucleotide sequences).Accordingly another embodiment of the invention relates to an isolatednucleic acid sequence comprising at least one transcription regulatingnucleotide sequence as described by SEQ ID NOs: 6, 7, 8, 11, 12, 13, 19,20, or 21.

In a more preferred embodiment the transcription regulating nucleotidesequence is selected from the group of sequences consisting of

-   i) the sequences described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12,    13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61,    62, 63, 66, 67, 68, 71, 72, and 73, and-   ii) a fragment of at least 50 (preferably at least 70 or 100, more    preferably at least 150 or 200, even more preferably at least 300 or    400, most preferably at least 500 or 700) consecutive bases of a    sequence under i); and-   iii) a nucleotide sequence having substantial similarity (preferably    with a sequence identity of at least 60%; more preferably measured    by the BLASTN program with the default parameters wordlength (W) of    11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a    comparison of both strands) to a transcription regulating nucleotide    sequence described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14,    15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63,    66, 67, 68, 71, 72, or 73; and-   iv) a nucleotide sequence capable of hybridizing to a transcription    regulating nucleotide sequence described by SEQ ID NOs: 1, 2, 3, 6,    7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29,    56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73 or the complement    thereof (preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄,    1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C.; more    preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM    EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., still more    preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM    EDTA at 50° C. with washing in 0.5×SSC, 0.1% SDS at 50° C., even    more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1    mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., most    preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM    EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.); and-   v) a nucleotide sequence capable of hybridizing to a nucleic acid    comprising 50 to 200 or more ((preferably at least 70 or 100, more    preferably at least 150 or 200, even more preferably at least 300 or    400, most preferably at least 500 or 700) consecutive nucleotides of    a transcription regulating nucleotide sequence described by SEQ ID    NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23,    24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73 or    the complement thereof; and-   vii) a nucleotide sequence which is the complement or reverse    complement of any of the previously mentioned nucleotide sequences    under i) to v).

Another preferred embodiment relates to an expression cassette forregulating expression in monocotyledonous plants comprising

-   a) at least one transcription regulating nucleotide sequence    functional in a monocotyledonous plant comprising at least one    sequence selected from the group of sequences consisting of    -   i) the sequences described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11,        12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57,        58, 61, 62, 63, 66, 67, 68, 71, 72, and 73, and    -   ii) a fragment of at least 50 (preferably at least 70 or 100,        more preferably at least 150 or 200, even more preferably at        least 300 or 400, most preferably at least 500 or 700)        consecutive bases of a sequence under i); and    -   iii) a nucleotide sequence having substantial similarity        (preferably with a sequence identity of at least 60%; more        preferably measured by the BLASTN program with the default        parameters wordlength (W) of 11, an expectation (E) of 10, a        cutoff of 100, M=5, N=−4, and a comparison of both strands) to a        transcription regulating nucleotide sequence described by SEQ ID        NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22,        23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72,        or 73; and    -   iv) a nucleotide sequence capable of hybridizing to a        transcription regulating nucleotide sequence described by SEQ ID        NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22,        23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72,        or 73 or the complement thereof (preferably in 7% sodium dodecyl        sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in        2×SSC, 0.1% SDS at 50° C.; more preferably in 7% sodium dodecyl        sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in        1×SSC, 0.1% SDS at 50° C., still more preferably in 7% sodium        dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with        washing in 0.5×SSC, 0.1% SDS at 50° C., even more preferably in        7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at        50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., most        preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM        EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.); and    -   v) a nucleotide sequence capable of hybridizing to a nucleic        acid comprising 50 to 200 or more (preferably at least 70 or        100, more preferably at least 150 or 200, even more preferably        at least 300 or 400, most preferably at least 500 or 700)        consecutive nucleotides of a transcription regulating nucleotide        sequence described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13,        14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61,        62, 63, 66, 67, 68, 71, 72, or 73 or the complement thereof; and    -   vi) a nucleotide sequence which is the complement or reverse        complement of any of the previously mentioned nucleotide        sequences under i) to v),    -   and-   b) at least one nucleic acid sequence which is heterologous in    relation to said transcription regulating sequence.

Preferably, the sequences specified under ii), iii), iv) v) and vi) inthe paragraphs above are capable to modify transcription in amonocotyledonous plant cell or organism. More preferably said sequencesspecified under ii), iii), iv) v) and vi) have substantially the sametranscription regulating activity as the transcription regulatingnucleotide sequence described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12,13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62,63, 66, 67, 68, 71, 72, or 73.

Also preferably the sequences specified under iii) above have a sequenceidentity of at least 60%, preferably 70% or 80%, more preferably 90% or95% to a sequence described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13,14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63,66, 67, 68, 71, 72, or 73, wherein the identity is preferably measuredby the BLASTN program with the default parameters wordlength (W) of 11,an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparisonof both strands.

Further preferably, the sequences specified under iv) or v) above arehybridizing under stringent conditions, preferably under mediumstringent conditions, most preferably under high stringent conditions(such as in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.) with the specifiedtarget sequence.

The activity of a specific transcription regulating nucleotide sequenceis considered substantially the same or equivalent if transcription isinitiated preferentially or specifically in the same tissue than theoriginal promoter (e.g., in root and kernel or constitutive in all ormost tissues) under otherwise identical conditions (i.e. in combinationwith the set of additional regulatory elements (e.g., introns,transcription terminator sequences and 5′-untranslated regions) and thesame nucleic acid sequence to be expressed in the same plant expressionsystem). Such expression profile is preferably demonstrated usingreporter genes operably linked to said transcription regulatingsequence. Preferred reporter genes (Schenborn 1999) in this context aregreen fluorescence protein (GFP) (Chuff 1996; Leffel 1997),chloramphenicol transferase, luciferase (Millar 1992), R-glucuronidaseor β-galactosidase. Especially preferred is R-glucuronidase (Jefferson1987). With respect to promoters with constitutive expression activity,the term “at most times” means a transcription regulating activity (asdemonstrated by an β-glucuronidase assays as described in the examplesbelow) preferably during at least 50%, preferably at least 70%, morepreferably at least 90% of the development cycle of a plant comprisingthe respective expression cassette stably integrated into itschromosomal DNA.

With respect to a constitutive transcription regulating nucleotidesequence (e.g., a constitutive promoter), the term “in most tissues”means a transcription regulating activity (as demonstrated by anβ-glucuronidase assays as described in the examples below) in tissueswhich together account to preferably at least 50%, preferably at least70%, more preferably at least 90% of the entire biomass of the a plantcomprising the respective expression cassette stably integrated into itschromosomal DNA.

Beside this the transcription regulating activity of a functionequivalent may vary from the activity of its parent sequence, especiallywith respect to expression level. The expression level may be higher orlower than the expression level of the parent sequence. Both derivationsmay be advantageous depending on the nucleic acid sequence of interestto be expressed. Preferred are such functional equivalent sequenceswhich—in comparison with its parent sequence—does not derivate from theexpression level of said parent sequence by more than 50%, preferably25%, more preferably 10% (as to be preferably judged by either mRNAexpression or protein (e.g., reporter gene) expression). Furthermorepreferred are equivalent sequences which demonstrate an increasedexpression in comparison to its parent sequence, preferably an increasemy at least 50%, more preferably by at least 100%, most preferably by atleast 500%.

Such functional equivalent of the transcription regulating nucleotidesequence may be obtained from other monocotyledonous plant species byusing the transcription regulating sequences described herein as probesto screen for homologous structural genes in other plants byhybridization under low, moderate or stringent hybridization conditions.Regions of the transcription regulating sequences of the presentinvention, which are conserved among species, can also be used as PCRprimers to amplify a segment from a species other than rice or maize,and that segment used as a hybridization probe (the latter approachpermitting higher stringency screening) or in a transcription assay todetermine promoter activity. Moreover, the transcription regulatingsequences could be employed to identify structurally related sequencesin a database using computer algorithms.

More specifically, based on the transcription regulating sequences ofthe present invention, orthologs may be identified or isolated from thegenome of any desired organism, preferably from another plant, accordingto well known techniques based on their sequence similarity to thetranscription regulating sequences of the invention, e.g.,hybridization, PCR or computer generated sequence comparisons. Forexample, all or a portion of a particular transcription regulatingnucleotide sequence of the invention is used as a probe that selectivelyhybridizes to other gene sequences present in a population of clonedgenomic DNA fragments (i.e., genomic libraries) from a chosen sourceorganism. Further, suitable genomic libraries may be prepared from anycell or tissue of an organism. Such techniques include hybridizationscreening of plated DNA libraries (either plaques or colonies; see,e.g., Sambrook 1989) and amplification by PCR using oligonucleotideprimers preferably corresponding to sequence domains conserved amongrelated polypeptide or subsequences of the nucleotide sequences providedherein (see, e.g., Innis 1990). These methods are particularly wellsuited to the isolation of gene sequences from organisms closely relatedto the organism from which the probe sequence is derived. Theapplication of these methods using the transcription regulatingsequences of the invention as probes is well suited for the isolation ofgene sequences from any source organism, preferably other plant species.In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the sequence of theinvention. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook et al. (1989). In general, sequencesthat hybridize to the sequences disclosed herein will have at least 40%to 50%, about 60% to 70% and even about 80% 85%, 90%, 95% to 98% or moreidentity with the disclosed sequences. That is, the sequence similarityof sequences may range, sharing at least about 40% to 50%, about 60% to70%, and even about 80%, 85%, 90%, 95% to 98% sequence similarity.

The nucleic acid molecules of the invention can also be identified by,for example, a search of known databases for genes encoding polypeptideshaving a specified amino acid sequence identity or DNA having aspecified nucleotide sequence identity. Methods of alignment ofsequences for comparison are well known in the art and are describedhereinabove.

Hence, the isolated nucleic acid molecules of the invention include theorthologs of the transcription regulating sequences disclosed herein,i.e., the corresponding nucleotide sequences in other (i.e. other thanthe host organism where the specific sequences disclosed herein arederived from) monocotyledonous plant organisms, preferably, e.g., cerealplants such as corn, wheat, rye, barley, oats, turfgrass, sorghum,millet, or other monocotyledonous plants such as sugarcane or banana. Anortholog or orthologous gene is a gene from a different species thatencodes a product having the same or similar function, e.g., catalyzingthe same reaction as a product encoded by a gene from a referenceorganism. Thus, an ortholog includes polypeptides having less than,e.g., 90% amino acid sequence identity, but which ortholog encodes apolypeptide having the same or similar function. Databases such GenBankmay be employed to identify sequences related to the maize or ricesequences disclosed herein, e.g., orthologs in other monocotyledonousplants such as wheat, barley, oats and others. Alternatively,recombinant DNA techniques such as hybridization or PCR may be employedto identify sequences related to the maize and rice sequences or toclone the equivalent sequences from different maize or rice DNAs.

The transcription regulating nucleotide sequences of the invention ortheir functional equivalents can be obtained or isolated from any plantor non-plant source, or produced synthetically by purely chemical means.Preferred sources include, but are not limited to the plants defined inthe DEFINITION section above.

Thus, another embodiment of the invention relates to a method foridentifying and/or isolating a transcription regulating nucleotidesequence from a monocotyledonous plant characterized that saididentification and/or isolation utilizes a nucleic acid sequenceencoding an amino acid sequence as described by SEQ ID NOs: 5, 10, 18,26, 31, 60, 65, 70, or 75, or a parts of said nucleic acid sequence.Preferred are nucleic acid sequences described by SEQ ID NOs: 4, 9, 17,25, 30, 59, 64, 69, or 74 or parts thereof. “Part” in this context meansa nucleic acid sequence of at least 15 bases preferably at least 25bases, more preferably at least 50 bases. The method can be based on(but is not limited to) the methods described above such as polymerasechain reaction, hybridization or database screening. Preferably, thismethod of the invention is based on a polymerase chain reaction, whereinsaid nucleic acid sequence or its part is utilized as oligonucleotideprimer. The person skilled in the art is aware of several methods toamplify and isolate the promoter of a gene starting from part of itscoding sequence (such as, for example, part of a cDNA). Such methods mayinclude but are not limited to method such as inverse PCR (“iPCR”) or“thermal asymmetric interlaced FOR” (“TAIL PCR”).

Still another embodiment of the invention relates to a method forproviding a transgenic expression cassette for heterologous expressionin monocotyledonous plants comprising the steps of:

-   I. isolating of a transcription regulating nucleotide sequence from    a monocotyledonous plant utilizing at least one nucleic acid    sequence or a part thereof, wherein said sequence is encoding a    polypeptide described by SEQ ID NOs: 5, 10, 18, 26, 31, 60, 65, 70,    or 75, or a part of at least 15 bases of said nucleic acid sequence,    and-   III. functionally linking said transcription regulating nucleotide    sequence to another nucleotide sequence of interest, which is    heterologous in relation to said transcription regulating nucleotide    sequence.

Preferably, the nucleic acid sequence employed for the isolationcomprises at least 15 base, preferably at least 25 bases, morepreferably at least 50 bases of a sequence described by SEQ ID NOs: 4,9, 17, 25, 30, 59, 64, 69, or 74 Preferably, the isolation of thetranscription regulating nucleotide sequence is realized by a polymerasechain reaction utilizing said nucleic acid sequence as a primer. Theoperable linkage can be realized by standard cloning method known in theart such as ligation-mediated cloning or recombination-mediated cloning.

For both of the above mentioned methods preferably the nucleotidesequence utilized for isolation of said transcription regulatingnucleotide sequence is encoding a polypeptide comprising

-   a1) at least one sequence motif of a monocotyledonous plant lactate    dehydrogenase protein selected from the group consisting of the    amino acid sequences

i) (SEQ ID NO: 76) SLSELGFDA, ii) (SEQ ID NO: 77) VIGAGNVGMA, iii)(SEQ ID NO: 78) IVTAGARQI, iv) (SEQ ID NO: 79) L(F/Y)RKIVP, v)(SEQ ID NO: 80) GFPASRV, vi) (SEQ ID NO: 81) RF(L/I)AEHL, vii)(SEQ ID NO: 82) QAYMVGEH, viii)  (SEQ ID NO: 83) ALEGIRRAV, and ix)(SEQ ID NO: 84) GYSVAS(L/I)A,

-   -   or

-   a2) at least one sequence motif of a monocotyledonous plant    caffeoyl-CaA-O-methyltransferase protein selected from the group    consisting of the amino acid sequences

x) (SEQ ID NO: 85) EQKTRHSE, xi) (SEQ ID NO: 86) L(I/L)KLIGAK, xii)(SEQ ID NO: 87) KTMEIGVY, xiii)  (SEQ ID NO: 88) HERL(L/M)KLV, xiv)(SEQ ID NO: 89) CQLPVGDG,  and xv) (SEQ ID NO: 90) TLCRRVK.

Preferably, the transcription regulating nucleotide sequences andpromoters of the invention include a consecutive stretch of about 25 to2,000, including 50 to 500 or 100 to 250, and up to 1,000 or 1,500,contiguous nucleotides, e.g., 40 to about 743, 60 to about 743, 125 toabout 743, 250 to about 743, 400 to about 743, 600 to about 743, of anyone of SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21,22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, and73, or the promoter orthologs thereof, which include the minimalpromoter region.

In a particular embodiment of the invention said consecutive stretch ofabout 25 to 2,000, including 50 to 500 or 100 to 250, and up to 1,000 or1,500, contiguous nucleotides, e.g., 40 to about 743, 60 to about 743,125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743,has at least 75%, preferably 80%, more preferably 90% and mostpreferably 95%, nucleic acid sequence identity with a correspondingconsecutive stretch of about 25 to 2,000, including 50 to 500 or 100 to250, and up to 1,000 or 1,500, contiguous nucleotides, e.g., 40 to about743, 60 to about 743, 125 to about 743, 250 to about 743, 400 to about743, 600 to about 743, of any one of SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11,12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61,62, 63, 66, 67, 68, 71, 72, and 73, or the promoter orthologs thereof,which include the minimal promoter region. The above defined stretch ofcontiguous nucleotides preferably comprises one or more promoter motifsselected from the group consisting of TATA box, GC-box, CAAT-box and atranscription start site.

The transcription regulating nucleotide sequences of the invention ortheir functional equivalents are capable of driving expression inmonocotyledonous plants of a coding sequence in a target cell,particularly in a plant cell. The promoter sequences and methodsdisclosed herein are useful in regulating expression in monocotyledonousplants, respectively, of any heterologous nucleotide sequence in a hostplant in order to vary the phenotype of that plant. These promoters canbe used with combinations of enhancer, upstream elements, and/oractivating sequences from the 5′ flanking regions of plant expressiblestructural genes. Similarly the upstream element can be used incombination with various plant promoter sequences.

The transcription regulating nucleotide sequences and promoters of theinvention are useful to modify the phenotype of a plant. Various changesin the phenotype of a transgenic plant are desirable, i.e., modifyingthe fatty acid composition in a plant, altering the amino acid contentof a plant, altering a plant's pathogen defense mechanism, and the like.These results can be achieved by providing expression of heterologousproducts or increased expression of endogenous products in plants.Alternatively, the results can be achieved by providing for a reductionof expression of one or more endogenous products, particularly enzymesor cofactors in the plant. These changes result in an alteration in thephenotype of the transformed plant.

Generally, the transcription regulating nucleotide sequences andpromoters of the invention may be employed to express a nucleic acidsegment that is operably linked to said promoter such as, for example,an open reading frame, or a portion thereof, an anti-sense sequence, asequence encoding for a double-stranded RNA sequence, or a transgene inplants.

An operable linkage may—for example—comprise an sequential arrangementof the transcription regulating nucleotide sequence of the invention(for example a sequence as described by SEQ ID NOs: 1, 2, 3, 6, 7, 8,11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58,61, 62, 63, 66, 67, 68, 71, 72, or 73) with a nucleic acid sequence tobe expressed, and—optionally—additional regulatory elements such as forexample polyadenylation or transcription termination elements,enhancers, introns etc, in a way that the transcription regulatingnucleotide sequence can fulfill its function in the process ofexpression the nucleic acid sequence of interest under the appropriateconditions. The term “appropriate conditions” mean preferably thepresence of the expression cassette in a plant cell. Preferred arearrangements, in which the nucleic acid sequence of interest to beexpressed is placed down-stream (i.e., in 3′-direction) of thetranscription regulating nucleotide sequence of the invention in a way,that both sequences are covalently linked. Optionally additionalsequences may be inserted inbetween the two sequences. Such sequencesmay be for example linker or multiple cloning sites. Furthermore,sequences can be inserted coding for parts of fusion proteins (in case afusion protein of the protein encoded by the nucleic acid of interest isintended to be expressed). Preferably, the distance between the nucleicacid sequence of interest to be expressed and the transcriptionregulating nucleotide sequence of the invention is not more than 200base pairs, preferably not more than 100 base pairs, more preferably nomore than 50 base pairs.

An operable linkage in relation to any expression cassette or of theinvention may be realized by various methods known in the art,comprising both in vitro and in vivo procedure. Thus, an expressioncassette of the invention or an vector comprising such expressioncassette may by realized using standard recombination and cloningtechniques well known in the art (see e.g., Maniatis 1989; Silhavy 1984;Ausubel 1987).

An expression cassette may also be assembled by inserting atranscription regulating nucleotide sequence of the invention (forexample a sequence as described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12,13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62,63, 66, 67, 68, 71, 72, or 73) into the plant genome. Such insertionwill result in an operable linkage to a nucleic acid sequence ofinterest which as such already existed in the genome. By the insertionthe nucleic acid of interest is expressed in the desired way (e.g.,root/kernel-preferentially or root/kernel-specific or constitutive) dueto the transcription regulating properties of the transcriptionregulating sequence. The insertion may be directed or by chance.Preferably the insertion is directed and realized by for examplehomologous recombination. By this procedure a natural promoter may beexchanged against the transcription regulating nucleotide sequence ofthe invention, thereby modifying the expression profile of an endogenousgene. The transcription regulating nucleotide sequence may also beinserted in a way, that antisense mRNA of an endogenous gene isexpressed, thereby inducing gene silencing.

Similar, a nucleic acid sequence of interest to be expressed may byinserted into a plant genome comprising the transcription regulatingnucleotide sequence in its natural genomic environment (i.e. linked toits natural gene) in a way that the inserted sequence becomes operablylinked to the transcription regulating sequence, thereby forming anexpression cassette of the invention.

The open reading frame to be linked to the transcription regulatingnucleotide sequence of the invention may be obtained from an insectresistance gene, a disease resistance gene such as, for example, abacterial disease resistance gene, a fungal disease resistance gene, aviral disease resistance gene, a nematode disease resistance gene, aherbicide resistance gene, a gene affecting grain composition orquality, a nutrient utilization gene, a mycotoxin reduction gene, a malesterility gene, a selectable marker gene, a screenable marker gene, anegative selectable marker, a positive selectable marker, a geneaffecting plant agronomic characteristics, i.e., yield, standability,and the like, or an environment or stress resistance gene, i.e., one ormore genes that confer herbicide resistance or tolerance, insectresistance or tolerance, disease resistance or tolerance (viral,bacterial, fungal, oomycete, or nematode), stress tolerance orresistance (as exemplified by resistance or tolerance to drought, heat,chilling, freezing, excessive moisture, salt stress, or oxidativestress), increased yields, food content and makeup, physical appearance,male sterility, drydown, standability, prolificacy, starch properties orquantity, oil quantity and quality, amino acid or protein composition,and the like. By “resistant” is meant a plant which exhibitssubstantially no phenotypic changes as a consequence of agentadministration, infection with a pathogen, or exposure to stress. By“tolerant” is meant a plant which, although it may exhibit somephenotypic changes as a consequence of infection, does not have asubstantially decreased reproductive capacity or substantially alteredmetabolism.

The transcription regulating sequences of the invention with aconstitutive expression profile may be advantageously used forexpressing a wide variety of genes including those which alter metabolicpathways, confer disease resistance, for protein production, e.g.,antibody production, or to improve nutrient uptake and the like.Constitutive promoters may be modified so as to be regulatable, e.g.,inducible. The genes and promoters described hereinabove can be used toidentify orthologous genes and their promoters which are also likelyexpressed in a particular tissue and/or development manner. Moreover,the orthologous promoters are useful to express linked open readingframes. In addition, by aligning the promoters of these orthologs, novelcis elements can be identified that are useful to generate syntheticpromoters.

The expression regulating nucleotide sequences specified above may beoptionally operably linked to other suitable regulatory sequences, e.g.,a transcription terminator sequence, operator, repressor binding site,transcription factor binding site and/or an enhancer.

The present invention further provides a recombinant vector containingthe expression cassette of the invention, and host cells comprising theexpression cassette or vector, e.g., comprising a plasmid. Theexpression cassette or vector may augment the genome of a transformedplant or may be maintained extra chromosomally. The expression cassetteor vector of the invention may be present in the nucleus, chloroplast,mitochondria and/or plastid of the cells of the plant. Preferably, theexpression cassette or vector of the invention is comprised in thechromosomal DNA of the plant nucleus. The present invention alsoprovides a transgenic plant prepared by this method, a seed from such aplant and progeny plants from such a plant including hybrids andinbreds. The expression cassette may be operatively linked to astructural gene, the open reading frame thereof, or a portion thereof.The expression cassette may further comprise a Ti plasmid and becontained in an Agrobacterium tumefaciens cell; it may be carried on amicroparticle, wherein the microparticle is suitable for ballistictransformation of a plant cell; or it may be contained in a plant cellor protoplast. Further, the expression cassette or vector can becontained in a transformed plant or cells thereof, and the plant may bea dicot or a monocot. In particular, the plant may be a dicotyledonousplant. Preferred transgenic plants are transgenic maize, soybean,barley, alfalfa, sunflower, canola, soybean, cotton, peanut, sorghum,tobacco, sugarbeet, rice, wheat, rye, turfgrass, millet, sugarcane,tomato, or potato.

The invention also provides a method of plant breeding, e.g., to preparea crossed fertile transgenic plant. The method comprises crossing afertile transgenic plant comprising a particular expression cassette ofthe invention with itself or with a second plant, e.g., one lacking theparticular expression cassette, to prepare the seed of a crossed fertiletransgenic plant comprising the particular expression cassette. The seedis then planted to obtain a crossed fertile transgenic plant. The plantmay be a monocot or a dicot. In a particular embodiment, the plant is adicotyledonous plant. The crossed fertile transgenic plant may have theparticular expression cassette inherited through a female parent orthrough a male parent. The second plant may be an inbred plant. Thecrossed fertile transgenic may be a hybrid. Also included within thepresent invention are seeds of any of these crossed fertile transgenicplants.

The transcription regulating sequences of the invention further comprisesequences which are complementary to one (hereinafter “test” sequence)which hybridizes under stringent conditions with a nucleic acid moleculeas described by SEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16,19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68,71, 72, or 73 as well as RNA which is transcribed from the nucleic acidmolecule. When the hybridization is performed under stringentconditions, either the test or nucleic acid molecule of invention ispreferably supported, e.g., on a membrane or DNA chip. Thus, either adenatured test or nucleic acid molecule of the invention is preferablyfirst bound to a support and hybridization is effected for a specifiedperiod of time at a temperature of, e.g., between 55 and 70° C., indouble strength citrate buffered saline (SC) containing 0.1% SDSfollowed by rinsing of the support at the same temperature but with abuffer having a reduced SC concentration. Depending upon the degree ofstringency required such reduced concentration buffers are typicallysingle strength SC containing 0.1% SDS, half strength SC containing 0.1%SDS and one-tenth strength SC containing 0.1% SDS. More preferablyhybridization is carried out under high stringency conditions (asdefined above).

Virtually any DNA composition may be used for delivery to recipientplant cells, e.g., dicotyledonous cells, to ultimately produce fertiletransgenic plants in accordance with the present invention. For example,DNA segments or fragments in the form of vectors and plasmids, or linearDNA segments or fragments, in some instances containing only the DNAelement to be expressed in the plant, and the like, may be employed. Theconstruction of vectors which may be employed in conjunction with thepresent invention will be known to those of skill of the art in light ofthe present disclosure (see, e.g., Sambrook 1989; Gelvin 1990).

Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes), BACs(bacterial artificial chromosomes) and DNA segments for use intransforming such cells will, of course, generally comprise the cDNA,gene or genes which one desires to introduce into the cells. These DNAconstructs can further include structures such as promoters, enhancers,polylinkers, or even regulatory genes as desired. The DNA segment,fragment or gene chosen for cellular introduction will often encode aprotein which will be expressed in the resultant recombinant cells, suchas will result in a screenable or selectable trait and/or which willimpart an improved phenotype to the regenerated plant.

However, this may not always be the case, and the present invention alsoencompasses transgenic plants incorporating non-expressed transgenes.

In certain embodiments, it is contemplated that one may wish to employreplication-competent viral vectors in monocot transformation. Suchvectors include, for example, wheat dwarf virus (WDV) “shuttle” vectors,such as pW1-11 and PW1-GUS (Ugaki 1991). These vectors are capable ofautonomous replication in maize cells as well as E. coli, and as suchmay provide increased sensitivity for detecting DNA delivered totransgenic cells. A replicating vector may also be useful for deliveryof genes flanked by DNA sequences from transposable elements such as Ac,Ds, or Mu. It has been proposed (Laufs 1990) that transposition of theseelements within the maize genome requires DNA replication. It is alsocontemplated that transposable elements would be useful for introducingDNA segments or fragments lacking elements necessary for selection andmaintenance of the plasmid vector in bacteria, e.g., antibioticresistance genes and origins of DNA replication. It is also proposedthat use of a transposable element such as Ac, Ds, or Mu would activelypromote integration of the desired DNA and hence increase the frequencyof stably transformed cells. The use of a transposable element such asAc, Ds, or Mu may actively promote integration of the DNA of interestand hence increase the frequency of stably transformed cells.Transposable elements may be useful to allow separation of genes ofinterest from elements necessary for selection and maintenance of aplasmid vector in bacteria or selection of a transformant. By use of atransposable element, desirable and undesirable DNA sequences may betransposed apart from each other in the genome, such that throughgenetic segregation in progeny, one may identify plants with either thedesirable undesirable DNA sequences.

The nucleotide sequence of interest linked to one or more of thetranscription regulating sequences of the invention can, for example,code for a ribosomal RNA, an anti-sense RNA or any other type of RNAthat is not translated into protein. In another preferred embodiment ofthe invention, said nucleotide sequence of interest is translated into aprotein product. The transcription regulating nucleotide sequence and/ornucleotide sequence of interest linked thereto may be of homologous orheterologous origin with respect to the plant to be transformed. Arecombinant DNA molecule useful for introduction into plant cellsincludes that which has been derived or isolated from any source, thatmay be subsequently characterized as to structure, size and/or function,chemically altered, and later introduced into plants. An example of anucleotide sequence or segment of interest “derived” from a source,would be a nucleotide sequence or segment that is identified as a usefulfragment within a given organism, and which is then chemicallysynthesized in essentially pure form. An example of such a nucleotidesequence or segment of interest “isolated” from a source, would benucleotide sequence or segment that is excised or removed from saidsource by chemical means, e.g., by the use of restriction endonucleases,so that it can be further manipulated, e.g., amplified, for use in theinvention, by the methodology of genetic engineering. Such a nucleotidesequence or segment is commonly referred to as “recombinant.”

Therefore a useful nucleotide sequence, segment or fragment of interestincludes completely synthetic DNA, semi-synthetic DNA, DNA isolated frombiological sources, and DNA derived from introduced RNA. Generally, theintroduced DNA is not originally resident in the plant genotype which isthe recipient of the DNA, but it is within the scope of the invention toisolate a gene from a given plant genotype, and to subsequentlyintroduce multiple copies of the gene into the same genotype, e.g., toenhance production of a given gene product such as a storage protein ora protein that confers tolerance or resistance to water deficit.

The introduced recombinant DNA molecule includes but is not limited to,DNA from plant genes, and non-plant genes such as those from bacteria,yeasts, animals or viruses. The introduced DNA can include modifiedgenes, portions of genes, or chimeric genes, including genes from thesame or different genotype. The term “chimeric gene” or “chimeric DNA”is defined as a gene or DNA sequence or segment comprising at least twoDNA sequences or segments from species which do not combine DNA undernatural conditions, or which DNA sequences or segments are positioned orlinked in a manner which does not normally occur in the native genome ofuntransformed plant.

The introduced recombinant DNA molecule used for transformation hereinmay be circular or linear, double-stranded or single-stranded.Generally, the DNA is in the form of chimeric DNA, such as plasmid DNA,that can also contain coding regions flanked by regulatory sequenceswhich promote the expression of the recombinant DNA present in theresultant plant. Generally, the introduced recombinant DNA molecule willbe relatively small, i.e., less than about 30 kb to minimize anysusceptibility to physical, chemical, or enzymatic degradation which isknown to increase as the size of the nucleotide molecule increases. Asnoted above, the number of proteins, RNA transcripts or mixtures thereofwhich is introduced into the plant genome is preferably preselected anddefined, e.g., from one to about 5-10 such products of the introducedDNA may be formed.

Two principal methods for the control of expression are known, viz.:overexpression and underexpression. Overexpression can be achieved byinsertion of one or more than one extra copy of the selected gene. Itis, however, not unknown for plants or their progeny, originallytransformed with one or more than one extra copy of a nucleotidesequence, to exhibit the effects of underexpression as well asoverexpression. For underexpression there are two principle methodswhich are commonly referred to in the art as “antisense downregulation”and “sense downregulation” (sense downregulation is also referred to as“cosuppression”). Generically these processes are referred to as “genesilencing”. Both of these methods lead to an inhibition of expression ofthe target gene.

Obtaining sufficient levels of transgene expression in the appropriateplant tissues is an important aspect in the production of geneticallyengineered crops. Expression of heterologous DNA sequences in a planthost is dependent upon the presence of an operably linked promoter thatis functional within the plant host. Choice of the promoter sequencewill determine when and where within the organism the heterologous DNAsequence is expressed.

It is specifically contemplated by the inventors that one couldmutagenize a promoter to potentially improve the utility of the elementsfor the expression of transgenes in plants. The mutagenesis of theseelements can be carried out at random and the mutagenized promotersequences screened for activity in a trial-by-error procedure.Alternatively, particular sequences which provide the promoter withdesirable expression characteristics, or the promoter with expressionenhancement activity, could be identified and these or similar sequencesintroduced into the sequences via mutation. It is further contemplatedthat one could mutagenize these sequences in order to enhance theirexpression of transgenes in a particular species.

The means for mutagenizing a DNA segment encoding a promoter sequence ofthe current invention are well known to those of skill in the art. Asindicated, modifications to promoter or other regulatory element may bemade by random, or site-specific mutagenesis procedures. The promoterand other regulatory element may be modified by altering their structurethrough the addition or deletion of one or more nucleotides from thesequence which encodes the corresponding unmodified sequences.

Mutagenesis may be performed in accordance with any of the techniquesknown in the art, such as, and not limited to, synthesizing anoligonucleotide having one or more mutations within the sequence of aparticular regulatory region. In particular, site-specific mutagenesisis a technique useful in the preparation of promoter mutants, throughspecific mutagenesis of the underlying DNA. The technique furtherprovides a ready ability to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 toabout 75 nucleotides or more in length is preferred, with about 10 toabout 25 or more residues on both sides of the junction of the sequencebeing altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephages are readily commercially available and their use is generallywell known to those skilled in the art. Double stranded plasmids alsoare routinely employed in site directed mutagenesis which eliminates thestep of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the promoter. An oligonucleotideprimer bearing the desired mutated sequence is prepared, generallysynthetically. This primer is then annealed with the single-strandedvector, and subjected to DNA polymerizing enzymes such as E. colipolymerase I Klenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation. This heteroduplex vector is then used totransform or transfect appropriate cells, such as E. coli cells, andcells are selected which include recombinant vectors bearing the mutatedsequence arrangement. Vector DNA can then be isolated from these cellsand used for plant transformation. A genetic selection scheme wasdevised by Kunkel et al. (1987) to enrich for clones incorporatingmutagenic oligonucleotides. Alternatively, the use of PCR withcommercially available thermostable enzymes such as Taq polymerase maybe used to incorporate a mutagenic oligonucleotide primer into anamplified DNA fragment that can then be cloned into an appropriatecloning or expression vector. The PCR-mediated mutagenesis procedures ofTomic et al. (1990) and Upender et al. (1995) provide two examples ofsuch protocols. A PCR employing a thermostable ligase in addition to athermostable polymerase also may be used to incorporate a phosphorylatedmutagenic oligonucleotide into an amplified DNA fragment that may thenbe cloned into an appropriate cloning or expression vector. Themutagenesis procedure described by Michael (1994) provides an example ofone such protocol.

The preparation of sequence variants of the selected promoter-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting, asthere are other ways in which sequence variants of DNA sequences may beobtained. For example, recombinant vectors encoding the desired promotersequence may be treated with mutagenic agents, such as hydroxylamine, toobtain sequence variants.

As used herein; the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” also is intended to refer to a process thatinvolves the template-dependent extension of a primer molecule. The termtemplate-dependent process refers to nucleic acid synthesis of an RNA ora DNA molecule wherein the sequence of the newly synthesized strand ofnucleic acid is dictated by the well-known rules of complementary basepairing (see, for example, Watson and Rarnstad, 1987). Typically, vectormediated methodologies involve the introduction of the nucleic acidfragment into a DNA or RNA vector, the clonal amplification of thevector, and the recovery of the amplified nucleic acid fragment.Examples of such methodologies are provided by U.S. Pat. No. 4,237,224.A number of template dependent processes are available to amplify thetarget sequences of interest present in a sample, such methods beingwell known in the art and specifically disclosed herein below.

Where a clone comprising a promoter has been isolated in accordance withthe instant invention, one may wish to delimit the essential promoterregions within the clone. One efficient, targeted means for preparingmutagenizing promoters relies upon the identification of putativeregulatory elements within the promoter sequence. This can be initiatedby comparison with promoter sequences known to be expressed in similartissue-specific or developmentally unique manner. Sequences which areshared among promoters with similar expression patterns are likelycandidates for the binding of transcription factors and are thus likelyelements which confer expression patterns. Confirmation of theseputative regulatory elements can be achieved by deletion analysis ofeach putative regulatory region followed by functional analysis of eachdeletion construct by assay of a reporter gene which is functionallyattached to each construct. As such, once a starting promoter sequenceis provided, any of a number of different deletion mutants of thestarting promoter could be readily prepared.

Functionally equivalent fragments of a transcription regulatingnucleotide sequence of the invention can also be obtained by removing ordeleting non-essential sequences without deleting the essential one.Narrowing the transcription regulating nucleotide sequence to itsessential, transcription mediating elements can be realized in vitro bytrial-and-arrow deletion mutations, or in silico using promoter elementsearch routines. Regions essential for promoter activity oftendemonstrate clusters of certain, known promoter elements. Such analysiscan be performed using available computer algorithms such as PLACE(“Plant Cis-acting Regulatory DNA Elements”; Higo 1999), the B10BASEdatabase “Transfac” (Biologische Datenbanken GmbH, Braunschweig;Wingender 2001) or the database PlantCARE (Lescot 2002).

Preferably, functional equivalent fragments of one of the transcriptionregulating sequences of the invention comprises at least 100 base pairs,preferably, at least 200 base pairs, more preferably at least 500 basepairs of a transcription regulating nucleotide sequence as described bySEQ ID NOs: 1, 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22,23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73.More preferably this fragment is starting from the 3′-end of theindicated sequences.

Especially preferred are equivalent fragments of transcriptionregulating sequences, which are obtained by deleting the region encodingthe 5′-untranslated region of the mRNA, thus only providing the(untranscribed) promoter region. The 5′-untranslated region can beeasily determined by methods known in the art (such as 5′-RACEanalysis). Accordingly, some of the transcription regulating sequencesof the invention are equivalent fragments of other sequences (see Table2 below).

TABLE 2 Relationship of transcription regulating sequences of theinvention Transcription regulating sequence Equivalent sequenceEquivalent fragment SEQ ID NO: 1 (1034 bp) SEQ ID NO: 66 (997 bp) SEQ IDNO: 2 (992 bp) SEQ ID NO: 3 (301 bp) SEQ ID NO: 67 (900 bp) SEQ ID NO:68 (301 bp) SEQ ID NO: 6 (797 bp) SEQ ID NO: 7 (766 bp) SEQ ID NO: 8(301 bp) SEQ ID NO: 11 (1182 bp) SEQ ID NO: 14 (1270 bp) SEQ ID NO: 12(1111 bp) SEQ ID NO: 71 (1028 bp) SEQ ID NO: 13 (301 bp) SEQ ID NO: 15(1191 bp) SEQ ID NO: 16 (301 bp) SEQ ID NO: 72 (954 bp) SEQ ID NO: 73(301 bp) SEQ ID NO: 19 (1060 bp) SEQ ID NO: 22 (1093 bp) SEQ ID NO: 20(946 bp) SEQ ID NO: 56 (1000 bp) SEQ ID NO: 21 (301 bp) SEQ ID NO: 61(1000 bp) SEQ ID NO: 23 (948 bp) SEQ ID NO: 24 (301 bp) SEQ ID NO: 57(945 bp) SEQ ID NO: 58 (301 bp) SEQ ID NO: 62 (719 bp) SEQ ID NO: 63(301 bp) SEQ ID NO: 27 (998 bp) SEQ ID NO: 28 (948 bp) SEQ ID NO: 29(301 bp)

As indicated above, deletion mutants, deletion mutants of the promoterof the invention also could be randomly prepared and then assayed. Withthis strategy, a series of constructs are prepared, each containing adifferent portion of the clone (a subclone), and these constructs arethen screened for activity. A suitable means for screening for activityis to attach a deleted promoter or intron construct, which contains adeleted segment to a selectable or screenable marker, and to isolateonly those cells expressing the marker gene. In this way, a number ofdifferent, deleted promoter constructs are identified which still retainthe desired, or even enhanced, activity. The smallest segment which isrequired for activity is thereby identified through comparison of theselected constructs. This segment may then be used for the constructionof vectors for the expression of exogenous genes.

An expression cassette of the invention may comprise further regulatoryelements. The term in this context is to be understood in the broadmeaning comprising all sequences which may influence construction orfunction of the expression cassette. Regulatory elements may for examplemodify transcription and/or translation in prokaryotic or eukaryoticorganism. In an preferred embodiment the expression cassette of theinvention comprised downstream (in 3′-direction) of the nucleic acidsequence to be expressed a transcription termination sequenceand—optionally additional regulatory elements—each operably liked to thenucleic acid sequence to be expressed (or the transcription regulatingsequence).

The expression profile of the expression cassettes of the invention maybe modulated depending on the combination of the transcriptionregulating nucleotide sequence with expression enhancing introns and/ortranscriptions termination sequences. This in a preferred embodiment theexpression cassette of the inventions comprises at least one additionalelement selected from the group consisting of

a) 5′-untranslated regions, andb) intron encoding sequences, andc) transcription termination sequences.

The intron encoding sequences are preferably encoding an expressionenhancing intron from a monocotyledonous plant. More preferably theintron sequence is an intron from an ubiquitin, actin or alcoholdehydrogenase gene. Preferably, this intron is inserted in theexpression construct in the 5′-untranslated region of the nucleic acidsequence, which should be expressed (i.e., between the transcriptionregulating nucleotide sequence and the protein coding sequence (openreading frame) or the nucleic acid sequence to be expressed).

Preferably, the 5′-untranslated region is from the same gene as thetranscription regulating sequences.

The transcription terminating sequence preferably also comprises asequence inducing polyadenylation. The transcription terminatingsequence may be heterologous with respect to the transcriptionregulating nucleotide sequence and/or the nucleic acid sequence to beexpressed, but may also be the natural transcription regulatingnucleotide sequence of the gene of said transcription regulatingnucleotide sequence and/or said nucleic acid sequence to be expressed.In one preferred embodiment of the invention the transcriptionregulating nucleotide sequence is the natural transcription regulatingnucleotide sequence of the gene of the transcription regulatingsequence. Preferably the transcription termination sequence is selectedfrom the group of sequences described by SEQ ID NOs: 32, 34, and 35.

Additional regulatory elements may comprise additional promoter, minimalpromoters, or promoter elements, which may modify the expressionregulating properties. For example the expression may be made dependingon certain stress factors such water stress, abscisin (Lam 1991) or heatstress (Schoffl 1989). Furthermore additional promoters or promoterelements may be employed, which may realized expression in otherorganisms (such as E. coli or Agrobacterium). Such regulatory elementscan be find in the promoter sequences or bacteria such as amy and SPO2or in the promoter sequences of yeast or fungal promoters (such as ADC1,MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, and ADH).

Furthermore, it is contemplated that promoters combining elements frommore than one promoter may be useful. For example, U.S. Pat. No.5,491,288 discloses combining a Cauliflower Mosaic Virus promoter with ahistone promoter. Thus, the elements from the promoters disclosed hereinmay be combined with elements from other promoters. Promoters which areuseful for plant transgene expression include those that are inducible,viral, synthetic, constitutive (Odell 1985), temporally regulated,spatially regulated, tissue-specific, and spatial-temporally regulated.

Where expression in specific tissues or organs is desired,tissue-specific promoters may be used. In contrast, where geneexpression in response to a stimulus is desired, inducible promoters arethe regulatory elements of choice. Where continuous expression isdesired throughout the cells of a plant, constitutive promoters areutilized. Additional regulatory sequences upstream and/or downstreamfrom the core promoter sequence may be included in expression constructsof transformation vectors to bring about varying levels of expression ofheterologous nucleotide sequences in a transgenic plant.

A variety of 5′ and 3′ transcriptional regulatory sequences areavailable for use in the present invention. Transcriptional terminatorsare responsible for the termination of transcription and correct mRNApolyadenylation. The 3′ nontranslated regulatory DNA sequence preferablyincludes from about 50 to about 1,000, more preferably about 100 toabout 1,000, nucleotide base pairs and contains plant transcriptionaland translational termination sequences. Appropriate transcriptionalterminators and those which are known to function in plants include theCaMV 35S terminator, the tml terminator, the nopaline synthaseterminator, the pea rbcS E9 terminator, the terminator for the T7transcript from the octopine synthase gene of Agrobacterium tumefaciens,and the 3′ end of the protease inhibitor I or II genes from potato ortomato, although other 3′ elements known to those of skill in the artcan also be employed. Alternatively, one also could use a gamma coixin,oleosin 3 or other terminator from the genus Coix.

Preferred 3′ elements include those from the nopaline synthase gene ofAgrobacterium tumefaciens (Bevan 1983), the terminator for the T7transcript from the octopine synthase gene of Agrobacterium tumefaciens,and the 3′ end of the protease inhibitor I or II genes from potato ortomato.

As the DNA sequence between the transcription initiation site and thestart of the coding sequence, i.e., the untranslated leader sequence,can influence gene expression, one may also wish to employ a particularleader sequence. Preferred leader sequences are contemplated to includethose which include sequences predicted to direct optimum expression ofthe attached gene, i.e., to include a preferred consensus leadersequence which may increase or maintain mRNA stability and preventinappropriate initiation of translation. The choice of such sequenceswill be known to those of skill in the art in light of the presentdisclosure. Sequences that are derived from genes that are highlyexpressed in plants will be most preferred.

Preferred regulatory elements also include the 5′-untranslated region,introns and the 3′-untranslated region of genes. Such sequences thathave been found to enhance gene expression in transgenic plants includeintron sequences (e.g., from Adh1, bronze1, actin1, actin 2 (WO00/760067), or the sucrose synthase intron; see: The Maize Handbook,Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)) andviral leader sequences (e.g., from TMV, MCMV and AMV; Gallie 1987). Forexample, a number of non-translated leader sequences derived fromviruses are known to enhance expression. Specifically, leader sequencesfrom Tobacco Mosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV),and Alfalfa Mosaic Virus (AMV) have been shown to be effective inenhancing expression (e.g., Gallie 1987; Skuzeski 1990). Other leadersknown in the art include but are not limited to: Picornavirus leaders,for example, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein 1989); Potyvirus leaders, for example, TEV leader (TobaccoEtch Virus); MDMV leader (Maize Dwarf Mosaic Virus); Humanimmunoglobulin heavy-chain binding protein (BiP) leader, (Macejak 1991);Untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4), (Jobling 1987; Tobacco mosaic virus leader (TMV), (Gallie1989; and Maize Chlorotic Mottle Virus leader (MCMV) (Lommel 1991. Seealso, Della-Cioppa 1987. Regulatory elements such as Adh intron 1(Callis 1987), sucrose synthase intron (Vasil 1989) or TMV omega element(Gallie 1989), may further be included where desired.

Especially preferred are the 5′-untranslated region, introns and the3′-untranslated region from genes selected from the group of genesconsisting of caffeoyl-CoA-O-methyltransferase genes, C8,7-sterolisomerase genes, hydroxyproline-rich glycoprotein (HRGP) genes, lactatedehydrogenase genes, chloroplast protein 12 like genes. More preferably,the 5′-untranslated region, introns and the 3′-untranslated regionutilized in an expression cassette of the invention is from a plant geneselected from the group of genes consisting of Oryza sativacaffeoyl-CoA-O-methyltransferase genes, Oryza sativa C8,7-sterolisomerase genes, Zea may hydroxyproline-rich glycoprotein (HRGP) genes,Zea mays lactate dehydrogenase genes, Oryza sativa chloroplast protein12 like genes and functional equivalents thereof.

Most preferred are the 5′-untranslated regions comprised at the 3′-endof the sequences described by SEQ ID NOs: 1, 6, 11, 14, 19, 22, and 27.Especially preferred are the sequences described by nucleotide 993 to1034 of SEQ ID NO: 1, nucleotide 767 to 797 of SEQ ID NO: 6, nucleotide1112 to 1182 of SEQ ID NO 11, nucleotide 1192 to 1270 of SEQ ID NO 14,nucleotide 947 to 1060 of SEQ ID NO: 19, nucleotide 949 to 1093 of SEQID NO: 22, and nucleotide 949 to 998 of SEQ ID NO: 27.

The intron encoding sequences is preferably encoding an expressionenhancing intron from a monocotyledonous plant. Preferably, this intronis inserted in the expression construct in the 5′-untranslated region ofthe nucleic acid sequence, which should be expressed (i.e., between thetranscription regulating nucleotide sequence and the protein codingsequence (open reading frame) or the nucleic acid sequence to beexpressed). Most preferred as intron sequences are:

-   a) the introns of the Zea mays ubiquitin gene, preferably intron I    thereof, most preferably the intron sequence as described by    nucleotide 1,082 to 2,091 of SEQ ID NO: 36,-   b) the introns of the rice actin gene, preferably intron I thereof,    most preferably the intron sequence as described by nucleotide 121    to 568 of the sequence described by GenBank Acc.-No.: X63830,-   c) the introns of the Zea mays alcohol dehydrogenase (adh) gene,    preferably intron 6 thereof, most preferably the intron sequence as    described by nucleotide 3,135 to 3,476 of the sequence described by    GenBank Acc.-No.: X04049,

The transcription terminating sequence preferably also comprises asequence inducing polyadenylation. The transcription terminatingsequence may be heterologous with respect to the transcriptionregulating nucleotide sequence and/or the nucleic acid sequence to beexpressed, but may also be the natural transcription regulatingnucleotide sequence of the gene of said transcription regulatingnucleotide sequence and/or said nucleic acid sequence to be expressed.In one preferred embodiment of the invention the transcriptionregulating nucleotide sequence is the natural transcription regulatingnucleotide sequence of the gene of the transcription regulatingsequence. Preferred as transcription termination sequences are thetranscription termination sequences from a plant gene selected from thegroup of genes consisting of Oryza sativacaffeoyl-CoA-O-methyltransferase genes, Oryza sativa C8,7-sterolisomerase genes, Zea may hydroxyproline-rich glycoprotein (HRGP) genes,Zea mays lactate dehydrogenase genes, Oryza sativa chloroplast protein12 like genes and functional equivalents thereof. Most preferred are thetranscription termination sequence of the Zea mays lactate dehydrogenasegene as described by SEQ ID NO: 32, of the Oryza sativacaffeoyl-CoA-O-methyltransferase gene as described by SEQ ID NO: 34, andof the Zea may hydroxyproline-rich glycoprotein (HRGP) gene as describedby SEQ ID NO: 35. By the combination of the transcription regulatingsequences with specific 5′-untranslated regions, introns, and/ortranscription termination sequences it is possible to modulate theexpression specificity, especially tissue specificity.

Promoter 5′-UTR Intron Terminator Tissue Specificity Oryza sativa ownZea mays Own all (constitutive) Caffeoyl-CoA-O- Ubiquitinmethyltransferase Oryza sativa own Zea mays NOS root (kernel,Caffeoyl-CoA-O- Ubiquitin pollen) methyltransferase Oryza sativa own Zeamays NOS root, kernel C-8,7-sterol- Ubiquitin isomerase Zea maize ownZea mays Own root, silk Hydroxy-proline- Ubiquitin (kernel: rich glyco-embryo) protein (HRGP) Zea maize own Zea mays NOS or own root, kernelLactate- Ubiquitin dehydrogenase Chloroplast own Zea mays NOS Leaf,protein 12 Ubiquitin endosperm lie protein own: element of the same geneto which the promoter naturally belongs; NOS: nopaline synthase.

Additional preferred regulatory elements are enhancer sequences orpolyadenylation sequences. Preferred polyadenylation sequences are thosefrom plant genes or Agrobacterium T-DNA genes (such as for example theterminator sequences of the OCS (octopine synthase) or NOS (nopalinesynthase) genes).

Examples of enhancers include elements from the CaMV 35S promoter,octopine synthase genes (Ellis et al., 1987), the rice actin I gene, themaize alcohol dehydrogenase gene (Callis 1987), the maize shrunken Igene (Vasil 1989), TMV Omega element (Gallie 1989) and promoters fromnon-plant eukaryotes (e.g. yeast; Ma 1988). Vectors for use inaccordance with the present invention may be constructed to include theocs enhancer element. This element was first identified as a 16 bppalindromic enhancer from the octopine synthase (ocs) gene of ultilane(Ellis 1987), and is present in at least 10 other promoters (Bouchez1989). The use of an enhancer element, such as the ocs elements andparticularly multiple copies of the element, will act to increase thelevel of transcription from adjacent promoters when applied in thecontext of plant transformation.

An expression cassette of the invention (or a vector derived therefrom)may comprise additional functional elements, which are to be understoodin the broad sense as all elements which influence construction,propagation, or function of an expression cassette or a vector or atransgenic organism comprising them. Such functional elements mayinclude origin of replications (to allow replication in bacteria; forthe ORI of pBR322 or the P15A ori; Sambrook 1989), or elements requiredfor Agrobacterium T-DNA transfer (such as for example the left and/orrights border of the T-DNA).

Ultimately, the most desirable DNA segments for introduction into, forexample, a monocotyledonous genome, may be homologous genes or genefamilies which encode a desired trait (e.g., increased yield per acre)and which are introduced under the control of novel promoters orenhancers, etc., or perhaps even homologous or tissue specific (e.g.,root-, collar/sheath-, whorl-, stalk-, earshank-, kernel- orleaf-specific) promoters or control elements. Indeed, it is envisionedthat a particular use of the present invention will be the expression ofa gene in a constitutive manner or a root/kernel-preferential orroot/kernel-specific manner.

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This will generally be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit or signal peptide willtransport the protein to a particular intracellular or extracellulardestination, respectively, and will then be post-translationallyremoved. Transit or signal peptides act by facilitating the transport ofproteins through intracellular membranes, e.g., vacuole, vesicle,plastid and mitochondrial membranes, whereas signal peptides directproteins through the extracellular membrane.

A particular example of such a use concerns the direction of a herbicideresistance gene, such as the EPSPS gene, to a particular organelle suchas the chloroplast rather than to the cytoplasm. This is exemplified bythe use of the rbcs transit peptide which confers plastid-specifictargeting of proteins. In addition, it is proposed that it may bedesirable to target certain genes responsible for male sterility to themitochondria, or to target certain genes for resistance tophytopathogenic organisms to the extracellular spaces, or to targetproteins to the vacuole.

By facilitating the transport of the protein into compartments insideand outside the cell, these sequences may increase the accumulation ofgene product protecting them from proteolytic degradation. Thesesequences also allow for additional mRNA sequences from highly expressedgenes to be attached to the coding sequence of the genes. Since mRNAbeing translated by ribosomes is more stable than naked mRNA, thepresence of translatable mRNA in front of the gene may increase theoverall stability of the mRNA transcript from the gene and therebyincrease synthesis of the gene product. Since transit and signalsequences are usually post-translationally removed from the initialtranslation product, the use of these sequences allows for the additionof extra translated sequences that may not appear on the finalpolypeptide. Targeting of certain proteins may be desirable in order toenhance the stability of the protein (U.S. Pat. No. 5,545,818).

It may be useful to target DNA itself within a cell. For example, it maybe useful to target introduced DNA to the nucleus as this may increasethe frequency of transformation. Within the nucleus itself it would beuseful to target a gene in order to achieve site-specific integration.For example, it would be useful to have a gene introduced throughtransformation replace an existing gene in the cell. Other elementsinclude those that can be regulated by endogenous or exogenous agents,e.g., by zinc finger proteins, including naturally occurring zinc fingerproteins or chimeric zinc finger proteins (see, e.g., U.S. Pat. No.5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO98/53058; WO 00/23464; WO 95/19431; and WO 98/54311) or myb-liketranscription factors. For example, a chimeric zinc finger protein mayinclude amino acid sequences which bind to a specific DNA sequence (thezinc finger) and amino acid sequences that activate (e.g., GAL 4sequences) or repress the transcription of the sequences linked to thespecific DNA sequence.

It is one of the objects of the present invention to provide recombinantDNA molecules comprising a nucleotide sequence according to theinvention operably linked to a nucleotide segment of interest.

A nucleotide segment of interest is reflective of the commercial marketsand interests of those involved in the development of the crop. Cropsand markets of interest changes, and as developing nations open up worldmarkets, new crops and technologies will also emerge. In addition, asthe understanding of agronomic traits and characteristics such as yieldand heterosis increase, the choice of genes for transformation willchange accordingly. General categories of nucleotides of interestinclude, for example, genes involved in information, such as zincfingers, those involved in communication, such as kinases, and thoseinvolved in housekeeping, such as heat shock proteins. More specificcategories of transgenes, for example, include genes encoding importanttraits for agronomics, insect resistance, disease resistance, herbicideresistance, sterility, grain characteristics, and commercial products.Genes of interest include, generally, those involved in starch, oil,carbohydrate, or nutrient metabolism, as well as those affecting kernelsize, sucrose loading, zinc finger proteins, see, e.g., U.S. Pat. No.5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO98/53058; WO 00/23464; WO 95/19431; and WO 98/54311, and the like.

One skilled in the art recognizes that the expression level andregulation of a transgene in a plant can vary significantly from line toline. Thus, one has to test several lines to find one with the desiredexpression level and regulation. Once a line is identified with thedesired regulation specificity of a chimeric Cre transgene, it can becrossed with lines carrying different inactive replicons or inactivetransgene for activation.

Other sequences which may be linked to the gene of interest, whichencodes a polypeptide, are those which can target to a specificorganelle, e.g., to the mitochondria, nucleus, or plastid, within theplant cell. Targeting can be achieved by providing the polypeptide withan appropriate targeting peptide sequence, such as a secretory signalpeptide (for secretion or cell wall or membrane targeting, a plastidtransit peptide, a chloroplast transit peptide, e.g., the chlorophylla/b binding protein, a mitochondrial target peptide, a vacuole targetingpeptide, or a nuclear targeting peptide, and the like. For example, thesmall subunit of ribulose bisphosphate carboxylase transit peptide, theEPSPS transit peptide or the dihydrodipicolinic acid synthase transitpeptide may be used. For examples of plastid organelle targetingsequences (see WO 00/12732). Plastids are a class of plant organellesderived from proplastids and include chloroplasts, leucoplasts,amyloplasts, and chromoplasts. The plastids are major sites ofbiosynthesis in plants. In addition to photosynthesis in thechloroplast, plastids are also sites of lipid biosynthesis, nitratereduction to ammonium, and starch storage. And while plastids containtheir own circular, genome, most of the proteins localized to theplastids are encoded by the nuclear genome and are imported into theorganelle from the cytoplasm.

Transgenes used with the present invention will often be genes thatdirect the expression of a particular protein or polypeptide product,but they may also be non-expressible DNA segments, e.g., transposonssuch as Ds that do no direct their own transposition. As used herein, an“expressible gene” is any gene that is capable of being transcribed intoRNA (e.g., mRNA, antisense RNA, etc.) or translated into a protein,expressed as a trait of interest, or the like, etc., and is not limitedto selectable, screenable or non-selectable marker genes. The inventionalso contemplates that, where both an expressible gene that is notnecessarily a marker gene is employed in combination with a marker gene,one may employ the separate genes on either the same or different DNAsegments for transformation. In the latter case, the different vectorsare delivered concurrently to recipient cells to maximizecotransformation.

The choice of the particular DNA segments to be delivered to therecipient cells will often depend on the purpose of the transformation.One of the major purposes of transformation of crop plants is to addsome commercially desirable, agronomically important traits to theplant. Such traits include, but are not limited to, herbicide resistanceor tolerance; insect resistance or tolerance; disease resistance ortolerance (viral, bacterial, fungal, nematode); stress tolerance and/orresistance, as exemplified by resistance or tolerance to drought, heat,chilling, freezing, excessive moisture, salt stress; oxidative stress;increased yields; food content and makeup; physical appearance; malesterility; drydown; standability; prolificacy; starch properties; oilquantity and quality; and the like. One may desire to incorporate one ormore genes conferring any such desirable trait or traits, such as, forexample, a gene or genes encoding pathogen resistance.

In certain embodiments, the present invention contemplates thetransformation of a recipient cell with more than one advantageoustransgene. Two or more transgenes can be supplied in a singletransformation event using either distinct transgene-encoding vectors,or using a single vector incorporating two or more gene codingsequences. For example, plasmids bearing the bar and aroA expressionunits in either convergent, divergent, or colinear orientation, areconsidered to be particularly useful. Further preferred combinations arethose of an insect resistance gene, such as a Bt gene, along with aprotease inhibitor gene such as pinII, or the use of bar in combinationwith either of the above genes. Of course, any two or more transgenes ofany description, such as those conferring herbicide, insect, disease(viral, bacterial, fungal, nematode) or drought resistance, malesterility, drydown, standability, prolificacy, starch properties, oilquantity and quality, or those increasing yield or nutritional qualitymay be employed as desired.

1. Exemplary Transgenes

The transcription regulating sequences of the invention are especiallyuseful for expression (preferably constitutive orroot/kernel-preferential or root/kernel-specific expression) inmonocotyledonous plants (as defined above in the DEFINITION section),especially in cereal plants such as corn, rice, wheat, rye, barley andoats. However, a use in other plants (e.g., dicotyledonous or gymnospermplants) and other tissues cannot be ruled out.

The transcription regulating nucleotide sequences and expressioncassettes of the invention may be employed for numerous expressionpurposes such as for example expression of a protein, or expression ofan antisense RNA, sense or double-stranded RNA. Preferably, expressionof the nucleic acid sequence confers to the plant an agronomicallyvaluable trait.

1.1. Herbicide Resistance

The genes encoding phosphinothricin acetyltransferase (bar and pat),glyphosate tolerant EPSP synthase genes, the glyphosate degradativeenzyme gene gox encoding glyphosate oxidoreductase, deh (encoding adehalogenase enzyme that inactivates dalapon), herbicide resistant(e.g., sulfonylurea and imidazolinone) acetolactate synthase, and bxngenes (encoding a nitrilase enzyme that degrades bromoxynil) are goodexamples of herbicide resistant genes for use in transformation. The barand pat genes code for an enzyme, phosphinothricin acetyltransferase(PAT), which inactivates the herbicide phosphinothricin and preventsthis compound from inhibiting glutamine synthetase enzymes. The enzyme5-enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase), is normallyinhibited by the herbicide N-(phosphonomethyl)glycine (glyphosate).However, genes are known that encode glyphosate-resistant EPSP Synthaseenzymes. The deh gene encodes the enzyme dalapon dehalogenase andconfers resistance to the herbicide dalapon. The bxn gene codes for aspecific nitrilase enzyme that converts bromoxynil to a non-herbicidaldegradation product.

1.2 Insect Resistance

An important aspect of the present invention concerns the introductionof insect resistance-conferring genes into plants. Potential insectresistance genes which can be introduced include Bacillus thuringiensiscrystal toxin genes or Bt genes (Watrud 1985). Bt genes may provideresistance to lepidopteran or coleopteran pests such as European CornBorer (ECB) and corn rootworm (CRW). Preferred Bt toxin genes for use insuch embodiments include the CryIA(b) and CryIA(c) genes. Endotoxingenes from other species of B. thuringiensis which affect insect growthor development may also be employed in this regard. Protease inhibitorsmay also provide insect resistance (Johnson 1989), and will thus haveutility in plant transformation. The use of a protease inhibitor IIgene, pinII, from tomato or potato is envisioned to be particularlyuseful. Even more advantageous is the use of a pinII gene in combinationwith a Bt toxin gene, the combined effect of which has been discoveredby the present inventors to produce synergistic insecticidal activity.Other genes which encode inhibitors of the insects' digestive system, orthose that encode enzymes or co-factors that facilitate the productionof inhibitors, may also be useful. This group may be exemplified bycystatin and amylase inhibitors, such as those from wheat and barley.

Also, genes encoding lectins may confer additional or alternativeinsecticide properties. Lectins (originally termed phytohemagglutinins)are multivalent carbohydrate-binding proteins which have the ability toagglutinate red blood cells from a range of species. Lectins have beenidentified recently as insecticidal agents with activity againstweevils, ECB and rootworm (Murdock 1990; Czapla & Lang, 1990). Lectingenes contemplated to be useful include, for example, barley and wheatgerm agglutinin (WGA) and rice lectins (Gatehouse 1984), with WGA beingpreferred.

Genes controlling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as, e.g.,lytic peptides, peptide hormones and toxins and venoms, form anotheraspect of the invention. For example, it is contemplated, that theexpression of juvenile hormone esterase, directed towards specificinsect pests, may also result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock 1990).

Transgenic plants expressing genes which encode enzymes that affect theintegrity of the insect cuticle form yet another aspect of theinvention. Such genes include those encoding, e.g., chitinase,proteases, lipases and also genes for the production of nikkomycin, acompound that inhibits chitin synthesis, the introduction of any ofwhich is contemplated to produce insect resistant maize plants. Genesthat code for activities that affect insect molting, such thoseaffecting the production of ecdysteroid UDP-glucosyl transferase, alsofall within the scope of the useful transgenes of the present invention.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host plant to insect pestsare also encompassed by the present invention. It may be possible, forinstance, to confer insecticidal activity on a plant by altering itssterol composition. Sterols are obtained by insects from their diet andare used for hormone synthesis and membrane stability. Thereforealterations in plant sterol composition by expression of novel genes,e.g., those that directly promote the production of undesirable sterolsor those that convert desirable sterols into undesirable forms, couldhave a negative effect on insect growth and/or development and henceendow the plant with insecticidal activity. Lipoxygenases are naturallyoccurring plant enzymes that have been shown to exhibit anti-nutritionaleffects on insects and to reduce the nutritional quality of their diet.Therefore, further embodiments of the invention concern transgenicplants with enhanced lipoxygenase activity which may be resistant toinsect feeding.

The present invention also provides methods and compositions by which toachieve qualitative or quantitative changes in plant secondarymetabolites. One example concerns transforming plants to produce DIMBOAwhich, it is contemplated, will confer resistance to European cornborer, rootworm and several other maize insect pests. Candidate genesthat are particularly considered for use in this regard include thosegenes at the bx locus known to be involved in the synthetic DIMBOApathway (Dunn 1981). The introduction of genes that can regulate theproduction of maysin, and genes involved in the production of dhurrin insorghum, is also contemplated to be of use in facilitating resistance toearworm and rootworm, respectively.

Tripsacum dactyloides is a species of grass that is resistant to certaininsects, including corn root worm. It is anticipated that genes encodingproteins that are toxic to insects or are involved in the biosynthesisof compounds toxic to insects will be isolated from Tripsacum and thatthese novel genes will be useful in conferring resistance to insects. Itis known that the basis of insect resistance in Tripsacum is genetic,because said resistance has been transferred to Zea mays via sexualcrosses (Branson & Guss, 1972).

Further genes encoding proteins characterized as having potentialinsecticidal activity may also be used as transgenes in accordanceherewith. Such genes include, for example, the cowpea trypsin inhibitor(CpTI; Hilder 1987) which may be used as a rootworm deterrent; genesencoding avermectin (Campbell 1989; Ikeda 1987) which may proveparticularly useful as a corn rootworm deterrent; ribosome inactivatingprotein genes; and even genes that regulate plant structures. Transgenicmaize including anti-insect antibody genes and genes that code forenzymes that can covert a non-toxic insecticide (pro-insecticide)applied to the outside of the plant into an insecticide inside the plantare also contemplated.

1.3 Environment or Stress Resistance

Improvement of a plant's ability to tolerate various environmentalstresses such as, but not limited to, drought, excess moisture,chilling, freezing, high temperature, salt, and oxidative stress, canalso be effected through expression of heterologous, or overexpressionof homologous genes. Benefits may be realized in terms of increasedresistance to freezing temperatures through the introduction of an“antifreeze” protein such as that of the Winter Flounder (Cutler 1989)or synthetic gene derivatives thereof. Improved chilling tolerance mayalso be conferred through increased expression of glycerol-3-phosphateacetyltransferase in chloroplasts (Murata 1992; Wolter 1992). Resistanceto oxidative stress (often exacerbated by conditions such as chillingtemperatures in combination with high light intensities) can beconferred by expression of superoxide dismutase (Gupta 1993), and may beimproved by glutathione reductase (Bowler 1992). Such strategies mayallow for tolerance to freezing in newly emerged fields as well asextending later maturity higher yielding varieties to earlier relativematurity zones.

Expression of novel genes that favorably effect plant water content,total water potential, osmotic potential, and turgor can enhance theability of the plant to tolerate drought. As used herein, the terms“drought resistance” and “drought tolerance” are used to refer to aplants increased resistance or tolerance to stress induced by areduction in water availability, as compared to normal circumstances,and the ability of the plant to function and survive in lower-waterenvironments, and perform in a relatively superior manner. In thisaspect of the invention it is proposed, for example, that the expressionof a gene encoding the biosynthesis of osmotically-active solutes canimpart protection against drought. Within this class of genes are DNAsencoding mannitol dehydrogenase (Lee and Saier, 1982) andtrehalose-6-phosphate synthase (Kaasen 1992). Through the subsequentaction of native phosphatases in the cell or by the introduction andcoexpression of a specific phosphatase, these introduced genes willresult in the accumulation of either mannitol or trehalose,respectively, both of which have been well documented as protectivecompounds able to mitigate the effects of stress. Mannitol accumulationin transgenic tobacco has been verified and preliminary results indicatethat plants expressing high levels of this metabolite are able totolerate an applied osmotic stress (Tarczynski 1992).

Similarly, the efficacy of other metabolites in protecting either enzymefunction (e.g. alanopine or propionic acid) or membrane integrity (e.g.,alanopine) has been documented (Loomis 1989), and therefore expressionof gene encoding the biosynthesis of these compounds can confer droughtresistance in a manner similar to or complimentary to mannitol. Otherexamples of naturally occurring metabolites that are osmotically activeand/or provide some direct protective effect during drought and/ordesiccation include sugars and sugar derivatives such as fructose,erythritol (Coxson 1992), sorbitol, dulcitol (Karsten 1992),glucosylglycerol (Reed 1984; Erdmann 1992), sucrose, stachyose (Koster &Leopold 1988; Blackman 1992), ononitol and pinitol (Vernon & Bohnert1992), and raffinose (Bernal-Lugo & Leopold 1992). Other osmoticallyactive solutes which are not sugars include, but are not limited to,proline and glycine-betaine (Wyn-Jones and Storey, 1981). Continuedcanopy growth and increased reproductive fitness during times of stresscan be augmented by introduction and expression of genes such as thosecontrolling the osmotically active compounds discussed above and othersuch compounds, as represented in one exemplary embodiment by the enzymemyoinositol-O-methyltransferase.

It is contemplated that the expression of specific proteins may alsoincrease drought tolerance. Three classes of Late Embryogenic Proteinshave been assigned based on structural similarities (see Dure 1989). Allthree classes of these proteins have been demonstrated in maturing(i.e., desiccating) seeds. Within these 3 types of proteins, the Type-II(dehydrin-type) have generally been implicated in drought and/ordesiccation tolerance in vegetative plant parts (e.g. Mundy and Chua,1988; Piatkowski 1990; Yamaguchi-Shinozaki 1992). Recently, expressionof a Type-III LEA (HVA-1) in tobacco was found to influence plantheight, maturity and drought tolerance (Fitzpatrick, 1993). Expressionof structural genes from all three groups may therefore confer droughttolerance. Other types of proteins induced during water stress includethiol proteases, aldolases and transmembrane transporters (Guerrero1990), which may confer various protective and/or repair-type functionsduring drought stress. The expression of a gene that effects lipidbiosynthesis and hence membrane composition can also be useful inconferring drought resistance on the plant.

Many genes that improve drought resistance have complementary modes ofaction. Thus, combinations of these genes might have additive and/orsynergistic effects in improving drought resistance in maize. Many ofthese genes also improve freezing tolerance (or resistance); thephysical stresses incurred during freezing and drought are similar innature and may be mitigated in similar fashion. Benefit may be conferredvia constitutive expression or root/kernel-preferential orroot/kernel-specific expression of these genes, but the preferred meansof expressing these novel genes may be through the use of aturgor-induced promoter (such as the promoters for the turgor-inducedgenes described in Guerrero et al. 1990 and Shagan 1993). Spatial andtemporal expression patterns of these genes may enable maize to betterwithstand stress.

Expression of genes that are involved with specific morphological traitsthat allow for increased water extractions from drying soil would be ofbenefit. For example, introduction and expression of genes that alterroot characteristics may enhance water uptake. Expression of genes thatenhance reproductive fitness during times of stress would be ofsignificant value. For example, expression of DNAs that improve thesynchrony of pollen shed and receptiveness of the female flower parts,i.e., silks, would be of benefit. In addition, expression of genes thatminimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value. Regulation ofcytokinin levels in monocots, such as maize, by introduction andexpression of an isopentenyl transferase gene with appropriateregulatory sequences can improve monocot stress resistance and yield(Gan 1995).

Given the overall role of water in determining yield, it is contemplatedthat enabling plants to utilize water more efficiently, through theintroduction and expression of novel genes, will improve overallperformance even when soil water availability is not limiting. Byintroducing genes that improve the ability of plants to maximize waterusage across a full range of stresses relating to water availability,yield stability or consistency of yield performance may be realized.

Improved protection of the plant to abiotic stress factors such asdrought, heat or chill, can also be achieved—for example—byoverexpressing antifreeze polypeptides from Myoxocephalus Scorpius (WO00/00512), Myoxocephalus octodecemspinosus, the Arabidopsis thalianatranscription activator CBF1, glutamate dehydrogenases (WO 97/12983, WO98/11240), calcium-dependent protein kinase genes (WO 98/26045),calcineurins (WO 99/05902), casein kinase from yeast (WO 02/052012),farnesyltransferases (WO 99/06580; Pei Z M et al. (1998) Science282:287-290), ferritin (Deak M et al. (1999) Nature Biotechnology17:192-196), oxalate oxidase (WO 99/04013; Dunwell J M (1998) BiotechnGenet Eng Rev 15:1-32), DREB1A factor (“dehydration response element B1A”; Kasuga M et al. (1999) Nature Biotech 17:276-286), genes ofmannitol or trehalose synthesis such as trehalose-phosphate synthase ortrehalose-phosphate phosphatase (WO 97/42326) or by inhibiting genessuch as trehalase (WO 97/50561).

1.4 Disease Resistance

It is proposed that increased resistance to diseases may be realizedthrough introduction of genes into plants period. It is possible toproduce resistance to diseases caused, by viruses, bacteria, fungi, rootpathogens, insects and nematodes. It is also contemplated that controlof mycotoxin producing organisms may be realized through expression ofintroduced genes.

Resistance to viruses may be produced through expression of novel genes.For example, it has been demonstrated that expression of a viral coatprotein in a transgenic plant can impart resistance to infection of theplant by that virus and perhaps other closely related viruses (Cuozzo1988, Hemenway 1988, Abel 1986). It is contemplated that expression ofantisense genes targeted at essential viral functions may impartresistance to said virus. For example, an antisense gene targeted at thegene responsible for replication of viral nucleic acid may inhibit saidreplication and lead to resistance to the virus. It is believed thatinterference with other viral functions through the use of antisensegenes may also increase resistance to viruses. Further it is proposedthat it may be possible to achieve resistance to viruses through otherapproaches, including, but not limited to the use of satellite viruses.

It is proposed that increased resistance to diseases caused by bacteriaand fungi may be realized through introduction of novel genes. It iscontemplated that genes encoding so-called “peptide antibiotics,”pathogenesis related (PR) proteins, toxin resistance, and proteinsaffecting host-pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences which are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in plants may beuseful in conferring resistance to bacterial disease. These genes areinduced following pathogen attack on a host plant and have been dividedinto at least five classes of proteins (Bol 1990). Included amongst thePR proteins are beta-1,3-glucanases, chitinases, and osmotin and otherproteins that are believed to function in plant resistance to diseaseorganisms. Other genes have been identified that have antifungalproperties, e.g., UDA (stinging nettle lectin) and hevein (Broakgert1989; Barkai-Golan 1978). It is known that certain plant diseases arecaused by the production of phytotoxins. Resistance to these diseasescould be achieved through expression of a novel gene that encodes anenzyme capable of degrading or otherwise inactivating the phytotoxin.Expression novel genes that alter the interactions between the hostplant and pathogen may be useful in reducing the ability the diseaseorganism to invade the tissues of the host plant, e.g., an increase inthe waxiness of the leaf cuticle or other morphological characteristics.

Plant parasitic nematodes are a cause of disease in many plants. It isproposed that it would be possible to make the plant resistant to theseorganisms through the expression of novel genes. It is anticipated thatcontrol of nematode infestations would be accomplished by altering theability of the nematode to recognize or attach to a host plant and/orenabling the plant to produce nematicidal compounds, including but notlimited to proteins.

Furthermore, a resistance to fungi, insects, nematodes and diseases, canbe achieved by targeted accumulation of certain metabolites or proteins.Such proteins include but are not limited to glucosinolates (defenseagainst herbivores), chitinases or glucanases and other enzymes whichdestroy the cell wall of parasites, ribosome-inactivating proteins(RIPs) and other proteins of the plant resistance and stress reaction asare induced when plants are wounded or attacked by microbes, orchemically, by, for example, salicylic acid, jasmonic acid or ethylene,or lysozymes from nonplant sources such as, for example, T4-lysozyme orlysozyme from a variety of mammals, insecticidal proteins such asBacillus thuringiensis endotoxin, a-amylase inhibitor or proteaseinhibitors (cowpea trypsin inhibitor), lectins such as wheatgermagglutinin, RNAses or ribozymes. Further examples are nucleic acidswhich encode the Trichoderma harzianum chit42 endochitinase (GenBankAcc. No.: S78423) or the N-hydroxylating, multi-functional cytochromeP-450 (CYP79) protein from Sorghum bicolor (GenBank Acc. No.: U32624),or functional equivalents of these. The accumulation of glucosinolatesas protection from pests (Rask L et al. (2000) Plant Mol Biol 42:93-113;Menard R et al. (1999) Phytochemistry 52:29-35), the expression ofBacillus thuringiensis endotoxins (Vaeck et al. (1987) Nature 328:33-37)or the protection against attack by fungi, by expression of chitinases,for example from beans (Broglie et al. (1991) Science 254:1194-1197), isadvantageous. Resistance to pests such as, for example, the rice pestNilaparvata lugens in rice plants can be achieved by expressing thesnowdrop (Galanthus nivalis) lectin agglutinin (Rao et al. (1998) PlantJ 15(4):469-77). The expression of synthetic cryIA(b) and cryIA(c)genes, which encode lepidoptera-specific Bacillus thuringiensisD-endotoxins can bring about a resistance to insect pests in variousplants (Goyal R K et al. (2000) Crop Protection 19(5):307-312). Furthertarget genes which are suitable for pathogen defense comprise“polygalacturonase-inhibiting protein” (PGIP), thaumatine, invertase andantimicrobial peptides such as lactoferrin (Lee T J et al. (2002) J AmerSoc Horticult Sci 127(2):158-164).

1.5 Mycotoxin Reduction/Elimination

Production of mycotoxins, including aflatoxin and fumonisin, by fungiassociated with plants is a significant factor in rendering the grainnot useful. These fungal organisms do not cause disease symptoms and/orinterfere with the growth of the plant, but they produce chemicals(mycotoxins) that are toxic to animals. Inhibition of the growth ofthese fungi would reduce the synthesis of these toxic substances and,therefore, reduce grain losses due to mycotoxin contamination. Novelgenes may be introduced into plants that would inhibit synthesis of themycotoxin without interfering with fungal growth. Expression of a novelgene which encodes an enzyme capable of rendering the mycotoxin nontoxicwould be useful in order to achieve reduced mycotoxin contamination ofgrain. The result of any of the above mechanisms would be a reducedpresence of mycotoxins on grain.

1.6 Grain Composition or Quality

Genes may be introduced into plants, particularly commercially importantcereals such as maize, wheat or rice, to improve the grain for which thecereal is primarily grown. A wide range of novel transgenic plantsproduced in this manner may be envisioned depending on the particularend use of the grain.

For example, the largest use of maize grain is for feed or food.Introduction of genes that alter the composition of the grain maygreatly enhance the feed or food value. The primary components of maizegrain are starch, protein, and oil. Each of these primary components ofmaize grain may be improved by altering its level or composition.Several examples may be mentioned for illustrative purposes but in noway provide an exhaustive list of possibilities.

The protein of many cereal grains is suboptimal for feed and foodpurposes especially when fed to pigs, poultry, and humans. The proteinis deficient in several amino acids that are essential in the diet ofthese species, requiring the addition of supplements to the grain.Limiting essential amino acids may include lysine, methionine,tryptophan, threonine, valine, arginine, and histidine. Some amino acidsbecome limiting only after the grain is supplemented with other inputsfor feed formulations. For example, when the grain is supplemented withsoybean meal to meet lysine requirements, methionine becomes limiting.The levels of these essential amino acids in seeds and grain may beelevated by mechanisms which include, but are not limited to, theintroduction of genes to increase the biosynthesis of the amino acids,decrease the degradation of the amino acids, increase the storage of theamino acids in proteins, or increase transport of the amino acids to theseeds or grain.

One mechanism for increasing the biosynthesis of the amino acids is tointroduce genes that deregulate the amino acid biosynthetic pathwayssuch that the plant can no longer adequately control the levels that areproduced. This may be done by deregulating or bypassing steps in theamino acid biosynthetic pathway which are normally regulated by levelsof the amino acid end product of the pathway. Examples include theintroduction of genes that encode deregulated versions of the enzymesaspartokinase or dihydrodipicolinic acid (DHDP)-synthase for increasinglysine and threonine production, and anthranilate synthase forincreasing tryptophan production. Reduction of the catabolism of theamino acids may be accomplished by introduction of DNA sequences thatreduce or eliminate the expression of genes encoding enzymes thatcatalyse steps in the catabolic pathways such as the enzymelysine-ketoglutarate reductase.

The protein composition of the grain may be altered to improve thebalance of amino acids in a variety of ways including elevatingexpression of native proteins, decreasing expression of those with poorcomposition, changing the composition of native proteins, or introducinggenes encoding entirely new proteins possessing superior composition.DNA may be introduced that decreases the expression of members of thezein family of storage proteins. This DNA may encode ribozymes orantisense sequences directed to impairing expression of zein proteins orexpression of regulators of zein expression such as the opaque-2 geneproduct. The protein composition of the grain may be modified throughthe phenomenon of cosuppression, i.e., inhibition of expression of anendogenous gene through the expression of an identical structural geneor gene fragment introduced through transformation (Goring 1991).Additionally, the introduced DNA may encode enzymes which degrade zeins.The decreases in zein expression that are achieved may be accompanied byincreases in proteins with more desirable amino acid composition orincreases in other major seed constituents such as starch.Alternatively, a chimeric gene may be introduced that comprises a codingsequence for a native protein of adequate amino acid composition such asfor one of the globulin proteins or 10 kD zein of maize and a promoteror other regulatory sequence designed to elevate expression of saidprotein. The coding sequence of said gene may include additional orreplacement codons for essential amino acids. Further, a coding sequenceobtained from another species, or, a partially or completely syntheticsequence encoding a completely unique peptide sequence designed toenhance the amino acid composition of the seed may be employed.

The introduction of genes that alter the oil content of the grain may beof value. Increases in oil content may result in increases inmetabolizable energy content and density of the seeds for uses in feedand food. The introduced genes may encode enzymes that remove or reducerate-limitations or regulated steps in fatty acid or lipid biosynthesis.Such genes may include, but are not limited to, those that encodeacetyl-CoA carboxylase, ACP-acyltransferase, beta-ketoacyl-ACP synthase,plus other well known fatty acid biosynthetic activities. Otherpossibilities are genes that encode proteins that do not possessenzymatic activity such as acyl carrier protein. Additional examplesinclude 2-acetyltransferase, oleosin pyruvate dehydrogenase complex,acetyl CoA synthetase, ATP citrate lyase, ADP-glucose pyrophosphorylaseand genes of the carnitine-CoA-acetyl-CoA shuttles. It is anticipatedthat expression of genes related to oil biosynthesis will be targeted tothe plastid, using a plastid transit peptide sequence and preferablyexpressed in the seed embryo. Genes may be introduced that alter thebalance of fatty acids present in the oil providing a more healthful ornutritive feedstuff. The introduced DNA may also encode sequences thatblock expression of enzymes involved in fatty acid biosynthesis,altering the proportions of fatty acids present in the grain such asdescribed below.

Genes may be introduced that enhance the nutritive value of the starchcomponent of the grain, for example by increasing the degree ofbranching, resulting in improved utilization of the starch in cows bydelaying its metabolism.

Besides affecting the major constituents of the grain, genes may beintroduced that affect a variety of other nutritive, processing, orother quality aspects of the grain as used for feed or food. Forexample, pigmentation of the grain may be increased or decreased.Enhancement and stability of yellow pigmentation is desirable in someanimal feeds and may be achieved by introduction of genes that result inenhanced production of xanthophylls and carotenes by eliminatingrate-limiting steps in their production. Such genes may encode alteredforms of the enzymes phytoene synthase, phytoene desaturase, or lycopenesynthase. Alternatively, unpigmented white corn is desirable forproduction of many food products and may be produced by the introductionof DNA which blocks or eliminates steps in pigment production pathways.

Feed or food comprising some cereal grains possesses insufficientquantities of vitamins and must be supplemented to provide adequatenutritive value. Introduction of genes that enhance vitamin biosynthesisin seeds may be envisioned including, for example, vitamins A, E,B.sub.12, choline, and the like. For example, maize grain also does notpossess sufficient mineral content for optimal nutritive value. Genesthat affect the accumulation or availability of compounds containingphosphorus, sulfur, calcium, manganese, zinc, and iron among otherswould be valuable. An example may be the introduction of a gene thatreduced phytic acid production or encoded the enzyme phytase whichenhances phytic acid breakdown. These genes would increase levels ofavailable phosphate in the diet, reducing the need for supplementationwith mineral phosphate.

Numerous other examples of improvement of cereals for feed and foodpurposes might be described. The improvements may not even necessarilyinvolve the grain, but may, for example, improve the value of the grainfor silage. Introduction of DNA to accomplish this might includesequences that alter lignin production such as those that result in the“brown midrib” phenotype associated with superior feed value for cattle.

In addition to direct improvements in feed or food value, genes may alsobe introduced which improve the processing of grain and improve thevalue of the products resulting from the processing. The primary methodof processing certain grains such as maize is via wetmilling. Maize maybe improved though the expression of novel genes that increase theefficiency and reduce the cost of processing such as by decreasingsteeping time.

Improving the value of wetmilling products may include altering thequantity or quality of starch, oil, corn gluten meal, or the componentsof corn gluten feed. Elevation of starch may be achieved through theidentification and elimination of rate limiting steps in starchbiosynthesis or by decreasing levels of the other components of thegrain resulting in proportional increases in starch. An example of theformer may be the introduction of genes encoding ADP-glucosepyrophosphorylase enzymes with altered regulatory activity or which areexpressed at higher level. Examples of the latter may include selectiveinhibitors of, for example, protein or oil biosynthesis expressed duringlater stages of kernel development.

The properties of starch may be beneficially altered by changing theratio of amylose to amylopectin, the size of the starch molecules, ortheir branching pattern. Through these changes a broad range ofproperties may be modified which include, but are not limited to,changes in gelatinization temperature, heat of gelatinization, clarityof films and pastes, Theological properties, and the like. To accomplishthese changes in properties, genes that encode granule-bound or solublestarch synthase activity or branching enzyme activity may be introducedalone or combination. DNA such as antisense constructs may also be usedto decrease levels of endogenous activity of these enzymes. Theintroduced genes or constructs may possess regulatory sequences thattime their expression to specific intervals in starch biosynthesis andstarch granule development. Furthermore, it may be advisable tointroduce and express genes that result in the in vivo derivatization,or other modification, of the glucose moieties of the starch molecule.The covalent attachment of any molecule may be envisioned, limited onlyby the existence of enzymes that catalyze the derivatizations and theaccessibility of appropriate substrates in the starch granule. Examplesof important derivations may include the addition of functional groupssuch as amines, carboxyls, or phosphate groups which provide sites forsubsequent in vitro derivatizations or affect starch properties throughthe introduction of ionic charges. Examples of other modifications mayinclude direct changes of the glucose units such as loss of hydroxylgroups or their oxidation to aldehyde or carboxyl groups.

Oil is another product of wetmilling of corn and other grains, the valueof which may be improved by introduction and expression of genes. Thequantity of oil that can be extracted by wetmilling may be elevated byapproaches as described for feed and food above. Oil properties may alsobe altered to improve its performance in the production and use ofcooking oil, shortenings, lubricants or other oil-derived products orimprovement of its health attributes when used in the food-relatedapplications. Novel fatty acids may also be synthesized which uponextraction can serve as starting materials for chemical syntheses. Thechanges in oil properties may be achieved by altering the type, level,or lipid arrangement of the fatty acids present in the oil. This in turnmay be accomplished by the addition of genes that encode enzymes thatcatalyze the synthesis of novel fatty acids and the lipids possessingthem or by increasing levels of native fatty acids while possiblyreducing levels of precursors. Alternatively DNA sequences may beintroduced which slow or block steps in fatty acid biosynthesisresulting in the increase in precursor fatty acid intermediates. Genesthat might be added include desaturases, epoxidases, hydratases,dehydratases, and other enzymes that catalyze reactions involving fattyacid intermediates. Representative examples of catalytic steps thatmight be blocked include the desaturations from stearic to oleic acidand oleic to linolenic acid resulting in the respective accumulations ofstearic and oleic acids.

Improvements in the other major cereal wetmilling products, gluten mealand gluten feed, may also be achieved by the introduction of genes toobtain novel plants. Representative possibilities include but are notlimited to those described above for improvement of food and feed value.

In addition it may further be considered that the plant be used for theproduction or manufacturing of useful biological compounds that wereeither not produced at all, or not produced at the same level, in theplant previously. The novel plants producing these compounds are madepossible by the introduction and expression of genes by transformationmethods. The possibilities include, but are not limited to, anybiological compound which is presently produced by any organism such asproteins, nucleic acids, primary and intermediary metabolites,carbohydrate polymers, etc. The compounds may be produced by the plant,extracted upon harvest and/or processing, and used for any presentlyrecognized useful purpose such as pharmaceuticals, fragrances,industrial enzymes to name a few.

Further possibilities to exemplify the range of grain traits orproperties potentially encoded by introduced genes in transgenic plantsinclude grain with less breakage susceptibility for export purposes orlarger grit size when processed by dry milling through introduction ofgenes that enhance gamma-zein synthesis, popcorn with improved popping,quality and expansion volume through genes that increase pericarpthickness, corn with whiter grain for food uses though introduction ofgenes that effectively block expression of enzymes involved in pigmentproduction pathways, and improved quality of alcoholic beverages orsweet corn through introduction of genes which affect flavor such as theshrunken gene (encoding sucrose synthase) for sweet corn.

1.7 Tuber or Seed Composition or Quality

Various traits can be advantageously expressed especially in seeds ortubers to improve composition or quality. Such traits include but arenot limited to:

-   -   Expression of metabolic enzymes for use in the food-and-feed        sector, for example of phytases and cellulases. Especially        preferred are nucleic acids such as the artificial cDNA which        encodes a microbial phytase (GenBank Acc. No.: A19451) or        functional equivalents thereof.    -   Expression of genes which bring about an accumulation of fine        chemicals such as of tocopherols, tocotrienols or carotenoids.        An example which may be mentioned is phytoene desaturase.        Preferred are nucleic acids which encode the Narcissus        pseudonarcissus photoene desaturase (GenBank Acc. No.: X78815)        or functional equivalents thereof.    -   Production of nutraceuticals such as, for example,        polyunsaturated fatty acids (for example arachidonic acid,        eicosapentaenoic acid or docosahexaenoic acid) by expression of        fatty acid elongases and/or desaturases, or production of        proteins with improved nutritional value such as, for example,        with a high content of essential amino acids (for example the        high-methionine 2S albumin gene of the brazil nut). Preferred        are nucleic acids which encode the Bertholletia excelsa        high-methionine 2S albumin (GenBank Acc. No.: AB044391), the        Physcomitrella patens 46-acyl-lipid desaturase (GenBank Acc.        No.: AJ222980; Girke et al. (1998) Plant J 15:39-48), the        Mortierella alpina 46-desaturase (Sakuradani et al. 1999 Gene        238:445-453), the Caenorhabditis elegans 45-desaturase        (Michaelson et al. 1998, FEBS Letters 439:215-218), the        Caenorhabditis elegans 45-fatty acid desaturase (des-5) (GenBank        Acc. No.: AF078796), the Mortierella alpina 45-desaturase        (Michaelson et al. JBC 273:19055-19059), the Caenorhabditis        elegans 46-elongase (Beaudoin et al. 2000, PNAS 97:6421-6426),        the Physcomitrella patens 46-elongase (Zank et al. 2000,        Biochemical Society Transactions 28:654-657), or functional        equivalents of these.    -   Production of high-quality proteins and enzymes for industrial        purposes (for example enzymes, such as lipases) or as        pharmaceuticals (such as, for example, antibodies, blood        clotting factors, interferons, lymphokins, colony stimulation        factor, plasminogen activators, hormones or vaccines, as        described by Hood E E, Jilka J M (1999) Curr Opin Biotechnol        10(4):382-6; Ma J K, Vine N D (1999) Curr Top Microbiol Immunol        236:275-92). For example, it has been possible to produce        recombinant avidin from chicken albumen and bacterial        β-glucuronidase (GUS) on a large scale in transgenic maize        plants (Hood et al. (1999) Adv Exp Med Biol 464:127-47. Review).    -   Obtaining an increased storability in cells which normally        comprise fewer storage proteins or storage lipids, with the        purpose of increasing the yield of these substances, for example        by expression of acetyl-CoA carboxylase. Preferred nucleic acids        are those which encode the Medicago sativa acetyl-CoA        carboxylase (ACCase) (GenBank Acc. No.: L25042), or functional        equivalents thereof.    -   Reducing levels of α-glucan L-type tuber phosphorylase (GLTP) or        α-glucan H-type tuber phosphorylase (GHTP) enzyme activity        preferably within the potato tuber (see U.S. Pat. No.        5,998,701). The conversion of starches to sugars in potato        tubers, particularly when stored at temperatures below 7° C., is        reduced in tubers exhibiting reduced GLTP or GHTP enzyme        activity. Reducing cold-sweetening in potatoes allows for potato        storage at cooler temperatures, resulting in prolonged dormancy,        reduced incidence of disease, and increased storage life.        Reduction of GLTP or GHTP activity within the potato tuber may        be accomplished by such techniques as suppression of gene        expression using homologous antisense or double-stranded RNA,        the use of co-suppression, regulatory silencing sequences. A        potato plant having improved cold-storage characteristics,        comprising a potato plant transformed with an expression        cassette having a TPT promoter sequence operably linked to a DNA        sequence comprising at least 20 nucleotides of a gene encoding        an α-glucan phosphorylase selected from the group consisting of        α-glucan L-type tuber phosphorylase (GLTP) and α-glucan H-type        phosphorylase (GHTP).

Further examples of advantageous genes are mentioned for example inDunwell J M, Transgenic approaches to crop improvement, J Exp Bot. 2000;51 Spec No; pages 487-96.

1.7 Plant Agronomic Characteristics

Two of the factors determining where plants can be grown are the averagedaily temperature during the growing season and the length of timebetween frosts. Within the areas where it is possible to grow aparticular plant, there are varying limitations on the maximal time itis allowed to grow to maturity and be harvested. The plant to be grownin a particular area is selected for its ability to mature and dry downto harvestable moisture content within the required period of time withmaximum possible yield. Therefore, plants of varying maturities aredeveloped for different growing locations. Apart from the need to drydown sufficiently to permit harvest is the desirability of havingmaximal drying take place in the field to minimize the amount of energyrequired for additional drying post-harvest. Also the more readily thegrain can dry down, the more time there is available for growth andkernel fill. Genes that influence maturity and/or dry down can beidentified and introduced into plant lines using transformationtechniques to create new varieties adapted to different growinglocations or the same growing location but having improved yield tomoisture ratio at harvest. Expression of genes that are involved inregulation of plant development may be especially useful, e.g., theliguleless and rough sheath genes that have been identified in plants.

Genes may be introduced into plants that would improve standability andother plant growth characteristics. For example, expression of novelgenes which confer stronger stalks, improved root systems, or prevent orreduce ear droppage would be of great value to the corn farmer.Introduction and expression of genes that increase the total amount ofphotoassimilate available by, for example, increasing light distributionand/or interception would be advantageous. In addition the expression ofgenes that increase the efficiency of photosynthesis and/or the leafcanopy would further increase gains in productivity. Such approacheswould allow for increased plant populations in the field.

Delay of late season vegetative senescence would increase the flow ofassimilate into the grain and thus increase yield. Overexpression ofgenes within plants that are associated with “stay green” or theexpression of any gene that delays senescence would be advantageous. Forexample, a non-yellowing mutant has been identified in Festuca pratensis(Davies 1990). Expression of this gene as well as others may preventpremature breakdown of chlorophyll and thus maintain canopy function.

1.8 Nutrient Utilization

The ability to utilize available nutrients and minerals may be alimiting factor in growth of many plants. It is proposed that it wouldbe possible to alter nutrient uptake, tolerate pH extremes, mobilizationthrough the plant, storage pools, and availability for metabolicactivities by the introduction of novel genes. These modifications wouldallow a plant to more efficiently utilize available nutrients. It iscontemplated that an increase in the activity of, for example, an enzymethat is normally present in the plant and involved in nutrientutilization would increase the availability of a nutrient. An example ofsuch an enzyme would be phytase. It is also contemplated that expressionof a novel gene may make a nutrient source available that was previouslynot accessible, e.g., an enzyme that releases a component of nutrientvalue from a more complex molecule, perhaps a macromolecule.

1.9 Male Sterility

Male sterility is useful in the production of hybrid seed. It isproposed that male sterility may be produced through expression of novelgenes. For example, it has been shown that expression of genes thatencode proteins that interfere with development of the maleinflorescence and/or gametophyte result in male sterility. Chimericribonuclease genes that express in the anthers of transgenic tobacco andoilseed rape have been demonstrated to lead to male sterility (Mariani1990). For example, a number of mutations were discovered in maize thatconfer cytoplasmic male sterility. One mutation in particular, referredto as T cytoplasm, also correlates with sensitivity to Southern cornleaf blight. A DNA sequence, designated TURF-13 (Levings 1990), wasidentified that correlates with T cytoplasm. It would be possiblethrough the introduction of TURF-13 via transformation to separate malesterility from disease sensitivity. As it is necessary to be able torestore male fertility for breeding purposes and for grain production,it is proposed that genes encoding restoration of male fertility mayalso be introduced.

1.10. Non-Protein-Expressing Sequences 1.10.1 RNA-Expressing

DNA may be introduced into plants for the purpose of expressing RNAtranscripts that function to affect plant phenotype yet are nottranslated into protein. Two examples are antisense RNA and RNA withribozyme activity. Both may serve possible functions in reducing oreliminating expression of native or introduced plant genes.

Genes may be constructed or isolated, which when transcribed, produceantisense RNA or double-stranded RNA that is complementary to all orpart(s) of a targeted messenger RNA(s). The antisense RNA reducesproduction of the polypeptide product of the messenger RNA. Thepolypeptide product may be any protein encoded by the plant genome. Theaforementioned genes will be referred to as antisense genes. Ananti-sense gene may thus be introduced into a plant by transformationmethods to produce a novel transgenic plant with reduced expression of aselected protein of interest. For example, the protein may be an enzymethat catalyzes a reaction in the plant. Reduction of the enzyme activitymay reduce or eliminate products of the reaction which include anyenzymatically synthesized compound in the plant such as fatty acids,amino acids, carbohydrates, nucleic acids and the like. Alternatively,the protein may be a storage protein, such as a zein, or a structuralprotein, the decreased expression of which may lead to changes in seedamino acid composition or plant morphological changes respectively. Thepossibilities cited above are provided only by way of example and do notrepresent the full range of applications.

Expression of antisense-RNA or double-stranded RNA by one of theexpression cassettes of the invention is especially preferred. Alsoexpression of sense RNA can be employed for gene silencing(co-suppression). This RNA is preferably a non-translatable RNA. Generegulation by double-stranded RNA (“double-stranded RNA interference”;dsRNAi) is well known in the art and described for various organismincluding plants (e.g., Matzke 2000; Fire A et al 1998; WO 99/32619; WO99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO00/63364).

Genes may also be constructed or isolated, which when transcribedproduce RNA enzymes, or ribozymes, which can act as endoribonucleasesand catalyze the cleavage of RNA molecules with selected sequences. Thecleavage of selected messenger RNA's can result in the reducedproduction of their encoded polypeptide products. These genes may beused to prepare novel transgenic plants which possess them. Thetransgenic plants may possess reduced levels of polypeptides includingbut not limited to the polypeptides cited above that may be affected byantisense RNA.

It is also possible that genes may be introduced to produce noveltransgenic plants which have reduced expression of a native gene productby a mechanism of cosuppression. It has been demonstrated in tobacco,tomato, and petunia (Goring 1991; Smith 1990; Napoli 1990; van der Krol1990) that expression of the sense transcript of a native gene willreduce or eliminate expression of the native gene in a manner similar tothat observed for antisense genes. The introduced gene may encode all orpart of the targeted native protein but its translation may not berequired for reduction of levels of that native protein.

1.10.2 Non-RNA-Expressing

For example, DNA elements including those of transposable elements suchas Ds, Ac, or Mu, may be, inserted into a gene and cause mutations.These DNA elements may be inserted in order to inactivate (or activate)a gene and thereby “tag” a particular trait. In this instance thetransposable element does not cause instability of the tagged mutation,because the utility of the element does not depend on its ability tomove in the genome. Once a desired trait is tagged, the introduced DNAsequence may be used to clone the corresponding gene, e.g., using theintroduced DNA sequence as a PCR primer together with PCR gene cloningtechniques (Shapiro, 1983; Dellaporta 1988). Once identified, the entiregene(s) for the particular trait, including control or regulatoryregions where desired may be isolated, cloned and manipulated asdesired. The utility of DNA elements introduced into an organism forpurposed of gene tagging is independent of the DNA sequence and does notdepend on any biological activity of the DNA sequence, i.e.,transcription into RNA or translation into protein. The sole function ofthe DNA element is to disrupt the DNA sequence of a gene.

It is contemplated that unexpressed DNA sequences, including novelsynthetic sequences could be introduced into cells as proprietary“labels” of those cells and plants and seeds thereof. It would not benecessary for a label DNA element to disrupt the function of a geneendogenous to the host organism, as the sole function of this DNA wouldbe to identify the origin of the organism. For example, one couldintroduce a unique DNA sequence into a plant and this DNA element wouldidentify all cells, plants, and progeny of these cells as having arisenfrom that labeled source. It is proposed that inclusion of label DNAswould enable one to distinguish proprietary germplasm or germplasmderived from such, from unlabelled germplasm.

Another possible element which may be introduced is a matrix attachmentregion element (MAR), such as the chicken lysozyme A element (Stief1989), which can be positioned around an expressible gene of interest toeffect an increase in overall expression of the gene and diminishposition dependant effects upon incorporation into the plant genome(Stief 1989; Phi-Van 1990).

Further nucleotide sequences of interest that may be contemplated foruse within the scope of the present invention in operable linkage withthe promoter sequences according to the invention are isolated nucleicacid molecules, e.g., DNA or RNA, comprising a plant nucleotide sequenceaccording to the invention comprising an open reading frame that ispreferentially expressed in a specific tissue, i.e., seed-, root, greentissue (leaf and stem), panicle-, or pollen, or is expressedconstitutively.

2. Marker Genes

In order to improve the ability to identify transformants, one maydesire to employ a selectable or screenable marker gene as, or inaddition to, the expressible gene of interest. “Marker genes” are genesthat impart a distinct phenotype to cells expressing the marker gene andthus allow such transformed cells to be distinguished from cells that donot have the marker. Such genes may encode either a selectable orscreenable marker, depending on whether the marker confers a trait whichone can ‘select’ for by chemical means, i.e., through the use of aselective agent (e.g., a herbicide, antibiotic, or the like), or whetherit is simply a trait that one can identify through observation ortesting, i.e., by ‘screening’ (e.g., the R-locus trait, the greenfluorescent protein (GFP)).

Of course, many examples of suitable marker genes are known to the artand can be employed in the practice of the invention.

Included within the terms selectable or screenable marker genes are alsogenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which encode a secretable antigen that can be identifiedby antibody interaction, or even secretable enzymes which can bedetected by their catalytic activity. Secretable proteins fall into anumber of classes, including small, diffusible proteins detectable,e.g., by ELISA; small active enzymes detectable in extracellularsolution (e.g., alpha-amylase, beta-lactamase, phosphinothricinacetyltransferase); and proteins that are inserted or trapped in thecell wall (e.g., proteins that include a leader sequence such as thatfound in the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

One example of a protein suitable for modification in this manner isextensin, or hydroxyproline-rich glycoprotein (HPRG). For example, themaize HPRG (Steifel 1990) molecule is well characterized in terms ofmolecular biology, expression and protein structure. However, any one ofa variety of ultilane and/or glycine-rich wall proteins (Keller 1989)could be modified by the addition of an antigenic site to create ascreenable marker.

One exemplary embodiment of a secretable screenable marker concerns theuse of a maize sequence encoding the wall protein HPRG, modified toinclude a 15 residue epitope from the pro-region of murine interleukin,however, virtually any detectable epitope may be employed in suchembodiments, as selected from the extremely wide variety ofantigen-antibody combinations known to those of skill in the art. Theunique extracellular epitope can then be straightforwardly detectedusing antibody labeling in conjunction with chromogenic or fluorescentadjuncts.

Elements of the present disclosure may be exemplified in detail throughthe use of the bar and/or GUS genes, and also through the use of variousother markers. Of course, in light of this disclosure, numerous otherpossible selectable and/or screenable marker genes will be apparent tothose of skill in the art in addition to the one set forth herein below.Therefore, it will be understood that the following discussion isexemplary rather than exhaustive. In light of the techniques disclosedherein and the general recombinant techniques which are known in theart, the present invention renders possible the introduction of anygene, including marker genes, into a recipient cell to generate atransformed plant.

2.1 Selectable Markers

Various selectable markers are known in the art suitable for planttransformation. Such markers may include but are not limited to:

2.1.1 Negative Selection Markers

Negative selection markers confer a resistance to a biocidal compoundsuch as a metabolic inhibitor (e.g., 2-deoxyglucose-6-phosphate, WO98/45456), antibiotics (e.g., kanamycin, G 418, bleomycin or hygromycin)or herbicides (e.g., phosphinothricin or glyphosate). Transformed plantmaterial (e.g., cells, tissues or plantlets), which express markergenes, are capable of developing in the presence of concentrations of acorresponding selection compound (e.g., antibiotic or herbicide) whichsuppresses growth of an untransformed wild type tissue. Especiallypreferred negative selection markers are those which confer resistanceto herbicides. Examples which may be mentioned are:

-   -   Phosphinothricin acetyltransferases (PAT; also named Bialophos®        resistance; bar; de Block 1987; Vasil 1992, 1993; Weeks 1993;        Becker 1994; Nehra 1994; Wan & Lemaux 1994; EP 0 333 033; U.S.        Pat. No. 4,975,374). Preferred are the bar gene from        Streptomyces hygroscopicus or the pat gene from Streptomyces        viridochromogenes. PAT inactivates the active ingredient in the        herbicide bialaphos, phosphinothricin (PPT). PPT inhibits        glutamine synthetase, (Murakami 1986; Twell 1989) causing rapid        accumulation of ammonia and cell death.    -   altered 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)        conferring resistance to Glyphosate®        (N-(phosphonomethyl)glycine) (Hinchee 1988; Shah 1986;        Della-Cioppa 1987). Where a mutant EPSP synthase gene is        employed, additional benefit may be realized through the        incorporation of a suitable chloroplast transit peptide, CTP        (EP-A1 0 218 571).    -   Glyphosate® degrading enzymes (Glyphosate® oxidoreductase; gox),    -   Dalapon® inactivating dehalogenases (deh)    -   sulfonylurea- and/or imidazolinone-inactivating acetolactate        synthases (ahas or ALS; for example mutated ahas/ALS variants        with, for example, the S4, X112, XA17, and/or Hra mutation        (EP-A1 154 204)    -   Bromoxynil® degrading nitrilases (bxn; Stalker 1988)    -   Kanamycin- or. geneticin (G418) resistance genes (NPTII; NPT or        neo; Potrykus 1985) coding e.g., for neomycin        phosphotransferases (Fraley 1983; Nehra 1994)    -   2-Desoxyglucose-6-phosphate phosphatase (DOG^(R)1-Gene product;        WO 98/45456; EP 0 807 836) conferring resistance against        2-desoxyglucose (Randez-Gil 1995).    -   hygromycin phosphotransferase (HPT), which mediates resistance        to hygromycin (Vanden Elzen 1985).    -   altered dihydrofolate reductase (Eichholtz 1987) conferring        resistance against methotrexat (Thillet 1988);    -   mutated anthranilate synthase genes that confers resistance to        5-methyl tryptophan.

Additional negative selectable marker genes of bacterial origin thatconfer resistance to antibiotics include the aadA gene, which confersresistance to the antibiotic spectinomycin, gentamycin acetyltransferase, streptomycin phosphotransferase (SPT),aminoglycoside-3-adenyl transferase and the bleomycin resistancedeterminant (Hayford 1988; Jones 1987; Svab 1990; Hille 1986).

Especially preferred are negative selection markers that conferresistance against the toxic effects imposed by D-amino acids like e.g.,D-alanine and D-serine (WO 03/060133; Erikson 2004). Especiallypreferred as negative selection marker in this contest are the dao1 gene(EC: 1.4.3.3: GenBank Acc.-No.: U60066) from the yeast Rhodotorulagracilis (Rhodosporidium toruloides) and the E. coli gene dsdA (D-serinedehydratase (D-serine deaminase) [EC: 4.3.1.18; GenBank Acc.-No.:J01603).

Transformed plant material (e.g., cells, embryos, tissues or plantlets)which express such marker genes are capable of developing in thepresence of concentrations of a corresponding selection compound (e.g.,antibiotic or herbicide) which suppresses growth of an untransformedwild type tissue. The resulting plants can be bred and hybridized in thecustomary fashion. Two or more generations should be grown in order toensure that the genomic integration is stable and hereditary.Corresponding methods are described (Jenes 1993; Potrykus 1991).

Furthermore, reporter genes can be employed to allow visual screening,which may or may not (depending on the type of reporter gene) requiresupplementation with a substrate as a selection compound.

Various time schemes can be employed for the various negative selectionmarker genes. In case of resistance genes (e.g., against herbicides orD-amino acids) selection is preferably applied throughout callusinduction phase for about 4 weeks and beyond at least 4 weeks intoregeneration. Such a selection scheme can be applied for all selectionregimes. It is furthermore possible (although not explicitly preferred)to remain the selection also throughout the entire regeneration schemeincluding rooting.

For example, with the phosphinotricin resistance gene (bar) as theselective marker, phosphinotricin at a concentration of from about 1 to50 mg/L may be included in the medium. For example, with the dao1 geneas the selective marker, D-serine or D-alanine at a concentration offrom about 3 to 100 mg/L may be included in the medium. Typicalconcentrations for selection are 20 to 40 mg/L. For example, with themutated ahas genes as the selective marker, PURSUIT™ at a concentrationof from about 3 to 100 mg/L may be included in the medium. Typicalconcentrations for selection are 20 to 40 mg/L.

2.1.2 Positive Selection Marker

Furthermore, positive selection marker can be employed. Genes likeisopentenyltransferase from Agrobacterium tumefaciens (strain:PO22;Genbank Acc.-No.: AB025109) may—as a key enzyme of the cytokininbiosynthesis—facilitate regeneration of transformed plants (e.g., byselection on cytokinin-free medium). Corresponding selection methods aredescribed (Ebinuma 2000a,b). Additional positive selection markers,which confer a growth advantage to a transformed plant in comparisonwith a non-transformed one, are described e.g., in EP-A 0 601 092.Growth stimulation selection markers may include (but shall not belimited to) β-Glucuronidase (in combination with e.g., a cytokininglucuronide), mannose-6-phosphate isomerase (in combination withmannose), UDP-galactose-4-epimerase (in combination with e.g.,galactose), wherein mannose-6-phosphate isomerase in combination withmannose is especially preferred.

2.1.3 Counter-Selection Marker

Counter-selection markers are especially suitable to select organismswith defined deleted sequences comprising said marker (Koprek 1999).Examples for counter-selection marker comprise thymidin kinases (TK),cytosine deaminases (Gleave 1999; Perera 1993; Stougaard 1993),cytochrom P450 proteins (Koprek 1999), haloalkan dehalogenases (Naested1999), iaaH gene products (Sundaresan 1995), cytosine deaminase codA(Schlaman & Hooykaas 1997), tms2 gene products (Fedoroff & Smith 1993),or α-naphthalene acetamide (NAM; Depicker 1988). Counter selectionmarkers may be useful in the construction of transposon tagging lines.For example, by marking an autonomous transposable element such as Ac,Master Mu, or En/Spn with a counter selection marker, one could selectfor transformants in which the autonomous element is not stablyintegrated into the genome. This would be desirable, for example, whentransient expression of the autonomous element is desired to activate intrans the transposition of a defective transposable element, such as Ds,but stable integration of the autonomous element is not desired. Thepresence of the autonomous element may not be desired in order tostabilize the defective element, i.e., prevent it from furthertransposing. However, it is proposed that if stable integration of anautonomous transposable element is desired in a plant the presence of anegative selectable marker may make it possible to eliminate theautonomous element during the breeding process.

2.2. Screenable Markers

Screenable markers that may be employed include, but are not limited to,a beta-glucuronidase (GUS) or uidA gene which encodes an enzyme forwhich various chromogenic substrates are known; an R-locus gene, whichencodes a product that regulates the production of anthocyanin pigments(red color) in plant tissues (Dellaporta 1988); a beta-lactamase gene(Sutcliffe 1978), which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylEgene (Zukowsky 1983) which encodes a catechol dioxygenase that canconvert chromogenic catechols; an α-amylase gene (Ikuta 1990); atyrosinase gene (Katz 1983) which encodes an enzyme capable of oxidizingtyrosine to DOPA and dopaquinone which in turn condenses to form theeasily detectable compound melanin; β-galactosidase gene, which encodesan enzyme for which there are chromogenic substrates; a luciferase (lux)gene (Ow 1986), which allows for bioluminescence detection; or even anaequorin gene (Prasher 1985), which may be employed in calcium-sensitivebioluminescence detection, or a green fluorescent protein gene (Niedz1995).

Genes from the maize R gene complex are contemplated to be particularlyuseful as screenable markers. The R gene complex in maize encodes aprotein that acts to regulate the production of anthocyanin pigments inmost seed and plant tissue. A gene from the R gene complex was appliedto maize transformation, because the expression of this gene intransformed cells does not harm the cells. Thus, an R gene introducedinto such cells will cause the expression of a red pigment and, ifstably incorporated, can be visually scored as a red sector. If a maizeline is carries dominant .quadrature.ultila for genes encoding theenzymatic intermediates in the anthocyanin biosynthetic pathway (C2, A1,A2, Bz1 and Bz2), but carries a recessive allele at the R locus,transformation of any cell from that line with R will result in redpigment formation. Exemplary lines include Wisconsin 22 which containsthe rg-Stadler allele and TR112, a K55 derivative which is r-g, b, P1.Alternatively any genotype of maize can be utilized if the C1 and Ralleles are introduced together.

It is further proposed that R gene regulatory regions may be employed inchimeric constructs in order to provide mechanisms for controlling theexpression of chimeric genes. More diversity of phenotypic expression isknown at the R locus than at any other locus (Coe 1988). It iscontemplated that regulatory regions obtained from regions 5′ to thestructural R gene would be valuable in directing the expression ofgenes, e.g., insect resistance, drought resistance, herbicide toleranceor other protein coding regions. For the purposes of the presentinvention, it is believed that any of the various R gene family membersmay be successfully employed (e.g., P, S, Lc, etc.). However, the mostpreferred will generally be Sn (particularly Sn:bol3). Sn is a dominantmember of the R gene complex and is functionally similar to the R and Bloci in that Sn controls the tissue specific deposition of anthocyaninpigments in certain seedling and plant cells, therefore, its phenotypeis similar to R.

A further screenable marker contemplated for use in the presentinvention is firefly luciferase, encoded by the lux gene. The presenceof the lux gene in transformed cells may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry. It is also envisioned that this system may be developed forpopulational screening for bioluminescence, such as on tissue cultureplates, or even for whole plant screening. Where use of a screenablemarker gene such as lux or GFP is desired, benefit may be realized bycreating a gene fusion between the screenable marker gene and aselectable marker gene, for example, a GFP-NPTII gene fusion. This couldallow, for example, selection of transformed cells followed by screeningof transgenic plants or seeds.

3. Exemplary DNA Molecules

The invention provides an isolated nucleic acid molecule, e.g., DNA orRNA, comprising a plant nucleotide sequence comprising an open readingframe that is preferentially expressed in a specific plant tissue (e.g.,roots and kernel) or is expressed constitutively, or a promoter thereof.

These promoters include, but are not limited to, constitutive,inducible, temporally regulated, developmentally regulated,spatially-regulated, chemically regulated, stress-responsive,tissue-specific, viral and synthetic promoters. Promoter sequences areknown to be strong or weak. A strong promoter provides for a high levelof gene expression, whereas a weak promoter provides for a very lowlevel of gene expression. An inducible promoter is a promoter thatprovides for the turning on and off of gene expression in response to anexogenously added agent, or to an environmental or developmentalstimulus. A bacterial promoter such as the P_(tac) promoter can beinduced to varying levels of gene expression depending on the level ofisothiopropylgalactoside added to the transformed bacterial cells. Anisolated promoter sequence that is a strong promoter for heterologousnucleic acid is advantageous because it provides for a sufficient levelof gene expression to allow for easy detection and selection oftransformed cells and provides for a high level of gene expression whendesired.

Within a plant promoter region there are several domains that arenecessary for full function of the promoter. The first of these domainslies immediately upstream of the structural gene and forms the “corepromoter region” containing consensus sequences, normally 70 base pairsimmediately upstream of the gene. The core promoter region contains thecharacteristic CAAT and TATA boxes plus surrounding sequences, andrepresents a transcription initiation sequence that defines thetranscription start point for the structural gene.

The presence of the core promoter region defines a sequence as being apromoter: if the region is absent, the promoter is non-functional.Furthermore, the core promoter region is insufficient to provide fullpromoter activity. A series of regulatory sequences upstream of the coreconstitute the remainder of the promoter. The regulatory sequencesdetermine expression level, the spatial and temporal pattern ofexpression and, for an important subset of promoters, expression underinductive conditions (regulation by external factors such as light,temperature, chemicals, hormones).

Regulated expression of the chimeric transacting viral replicationprotein can be further regulated by other genetic strategies. Forexample, Cre-mediated gene activation as described by Odell et al. 1990.Thus, a DNA fragment containing 3′ regulatory sequence bound by loxsites between the promoter and the replication protein coding sequencethat blocks the expression of a chimeric replication gene from thepromoter can be removed by Cre-mediated excision and result in theexpression of the trans-acting replication gene. In this case, thechimeric Cre gene, the chimeric trans-acting replication gene, or bothcan be under the control of tissue- and developmental-specific orinducible promoters. An alternate genetic strategy is the use of tRNAsuppressor gene. For example, the regulated expression of a tRNAsuppressor gene can conditionally control expression of a trans-actingreplication protein coding sequence containing an appropriatetermination codon as described by Ulmasov et al. 1997. Again, either thechimeric tRNA suppressor gene, the chimeric transacting replicationgene, or both can be under the control of tissue- anddevelopmental-specific or inducible promoters.

Frequently it is desirable to have continuous or inducible expression ofa DNA sequence throughout the cells of an organism in atissue-independent manner. For example, increased resistance of a plantt6 infection by soil- and airborne-pathogens might be accomplished bygenetic manipulation of the plant's genome to comprise a continuouspromoter operably linked to a heterologous pathogen-resistance gene suchthat pathogen-resistance proteins are continuously expressed throughoutthe plant's tissues.

Alternatively, it might be desirable to inhibit expression of a nativeDNA sequence within a plant's tissues to achieve a desired phenotype. Inthis case, such inhibition might be accomplished with transformation ofthe plant to comprise a constitutive, tissue-independent promoteroperably linked to an antisense nucleotide sequence, such thatconstitutive expression of the antisense sequence produces an RNAtranscript that interferes with translation of the mRNA of the nativeDNA sequence.

To define a minimal promoter region, a DNA segment representing thepromoter region is removed from the 5′ region of the gene of interestand operably linked to the coding sequence of a marker (reporter) geneby recombinant DNA techniques well known to the art. The reporter geneis operably linked downstream of the promoter, so that transcriptsinitiating at the promoter proceed through the reporter gene. Reportergenes generally encode proteins which are easily measured, including,but not limited to, chloramphenicol acetyl transferase (CAT),β-glucuronidase (GUS), green fluorescent protein (GFP), β-galactosidase(β-GAL), and luciferase.

The construct containing the reporter gene under the control of thepromoter is then introduced into an appropriate cell type bytransfection techniques well known to the art. To assay for the reporterprotein, cell lysates are prepared and appropriate assays, which arewell known in the art, for the reporter protein are performed. Forexample, if CAT were the reporter gene of choice, the lysates from cellstransfected with constructs containing CAT under the control of apromoter under study are mixed with isotopically labeled chloramphenicoland acetyl-coenzyme A (acetyl-CoA). The CAT enzyme transfers the acetylgroup from acetyl-CoA to the 2- or 3-position of chloramphenicol. Thereaction is monitored by thin-layer chromatography, which separatesacetylated chloramphenicol from unreacted material. The reactionproducts are then visualized by autoradiography.

The level of enzyme activity corresponds to the amount of enzyme thatwas made, which in turn reveals the level of expression from thepromoter of interest. This level of expression can be compared to otherpromoters to determine the relative strength of the promoter understudy. In order to be sure that the level of expression is determined bythe promoter, rather than by the stability of the mRNA, the level of thereporter mRNA can be measured directly, such as by Northern blotanalysis.

Once activity is detected, mutational and/or deletion analyses may beemployed to determine the minimal region and/or sequences required toinitiate transcription. Thus, sequences can be deleted at the 5′ end ofthe promoter region and/or at the 3′ end of the promoter region, andnucleotide substitutions introduced. These constructs are thenintroduced to cells and their activity determined.

In one embodiment, the promoter may be a gamma zein promoter, an oleosinole16 promoter, a globulins promoter, an actin I promoter, an actin cIpromoter, a sucrose synthetase promoter, an INOPS promoter, an EXM5promoter, a globulin2 promoter, a b-32, ADPG-pyrophosphorylase promoter,an LtpI promoter, an Ltp2 promoter, an oleosin ole17 promoter, anoleosin ole18 promoter, an actin 2 promoter, a pollen-specific proteinpromoter, a pollen-specific pectate lyase promoter, an anther-specificprotein promoter, an anther-specific gene RTS2 promoter, apollen-specific gene promoter, a tapetum-specific gene promoter,tapetum-specific gene RAB24 promoter, an a nthranilate synthase alphasubunit promoter, an alpha zein promoter, an anthranilate synthase betasubunit promoter, a dihydrodipicolinate synthase promoter, a Thilpromoter, an alcohol dehydrogenase promoter, a cab binding proteinpromoter, an H3C4 promoter, a RUBISCO SS starch branching enzymepromoter, an ACCase promoter, an actin3 promoter, an actin7 promoter, aregulatory protein GF14-12 promoter, a ribosomal protein L9 promoter, acellulose biosynthetic enzyme promoter, an S-adenosyl-L-homocysteinehydrolase promoter, a superoxide dismutase promoter, a C-kinase receptorpromoter, a phosphoglycerate mutase promoter, a root-specific RCc3 mRNApromoter, a glucose-6 phosphate isomerase promoter, apyrophosphate-fructose 6-phosphatelphosphotransferase promoter, anubiquitin promoter, a beta-ketoacyl-ACP synthase promoter, a 33 kDaphotosystem 11 promoter, an oxygen evolving protein promoter, a 69 kDavacuolar ATPase subunit promoter, a metallothionein-like proteinpromoter, a glyceraldehyde-3-phosphate dehydrogenase promoter, an ABA-and ripening-inducible-like protein promoter, a phenylalanine ammonialyase promoter, an adenosine triphosphatase S-adenosyl-L-homocysteinehydrolase promoter, an a-tubulin promoter, a cab promoter, a PEPCasepromoter, an R gene promoter, a lectin promoter, a light harvestingcomplex promoter, a heat shock protein promoter, a chalcone synthasepromoter, a zein promoter, a globulin-1 promoter, an ABA promoter, anauxin-binding protein promoter, a UDP glucose flavonoidglycosyl-transferase gene promoter, an NTI promoter, an actin promoter,an opaque 2 promoter, a b70 promoter, an oleosin promoter, a CaMV 35Spromoter, a CaMV 34S promoter, a CaMV 19S promoter, a histone promoter,a turgor-inducible promoter, a pea small subunit RuBP carboxylasepromoter, a Ti plasmid mannopine synthase promoter, Ti plasmid nopalinesynthase promoter, a petunia chalcone isomerase promoter, a bean glycinerich protein I promoter, a CaMV 35S transcript promoter, a potatopatatin promoter, or a S-E9 small subunit RuBP carboxylase promoter.

4. Transformed (Transgenic) Plants of the Invention and Methods ofPreparation

Plant species may be transformed with the DNA construct of the presentinvention by the DNA-mediated transformation of plant cell protoplastsand subsequent regeneration of the plant from the transformedprotoplasts in accordance with procedures well known in the art.

Any plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a vector of thepresent invention. The term “organogenesis,” as used herein, means aprocess by which shoots and roots are developed sequentially frommeristematic centers; the term “embryogenesis,” as used herein, means aprocess by which shoots and roots develop together in a concertedfashion (not sequentially), whether from somatic cells or gametes. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristems, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand ultilane meristem).

Plants of the present invention may take a variety of forms. The plantsmay be chimeras of transformed cells and non-transformed cells; theplants may be clonal transformants (e.g., all cells transformed tocontain the expression cassette); the plants may comprise grafts oftransformed and untransformed tissues (e.g., a transformed root stockgrafted to an untransformed scion in citrus species). The transformedplants may be propagated by a variety of means, such as by clonalpropagation or classical breeding techniques. For example, firstgeneration (or T1) transformed plants may be selfed to give homozygoussecond generation (or T2) transformed plants, and the T2 plants furtherpropagated through classical breeding techniques. A dominant selectablemarker (such as nptII) can be associated with the expression cassette toassist in breeding.

Thus, the present invention provides a transformed (transgenic) plantcell, in planta or ex planta, including a transformed plastid or otherorganelle, e.g., nucleus, mitochondria or chloroplast. The presentinvention may be used for transformation of any plant species,including, but not limited to, cells from the plant species specifiedabove in the DEFINITION section. Preferably, transgenic plants of thepresent invention are crop plants and in particular cereals (forexample, corn, alfalfa, sunflower, rice, Brassica, canola, soybean,barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.), and even more preferably corn, rice and soybean.Other embodiments of the invention are related to cells, cell cultures,tissues, parts (such as plants organs, leaves, roots, etc.) andpropagation material (such as seeds) of such plants.

The transgenic expression cassette of the invention may not only becomprised in plants or plant cells but may advantageously also becontaining in other organisms such for example bacteria. Thus, anotherembodiment of the invention relates to transgenic cells or non-human,transgenic organisms comprising an expression cassette of the invention.Preferred are prokaryotic and eukaryotic organism. Both microorganismand higher organisms are comprised. Preferred microorganisms arebacteria, yeast, algae, and fungi. Preferred bacteria are those of thegenus Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes,Pseudomonas, Bacillus or Cyanobacterim such as—for example—Synechocystisand other bacteria described in Brock Biology of Microorganisms EighthEdition (pages A-8, A-9, A10 and A11).

Especially preferred are microorganisms capable to infect plants and totransfer DNA into their genome, especially bacteria of the genusAgrobacterium, preferably Agrobacterium tumefaciens and rhizogenes.Preferred yeasts are Candida, Saccharomyces, Hansenula and Pichia.Preferred Fungi are Aspergillus, Trichoderma, Ashbya, Neurospora,Fusarium, and Beauveria. Most preferred are plant organisms as definedabove.

Transformation of plants can be undertaken with a single DNA molecule ormultiple DNA molecules (i.e., co-transformation), and both thesetechniques are suitable for use with the expression cassettes of thepresent invention. Numerous transformation vectors are available forplant transformation, and the expression cassettes of this invention canbe used in conjunction with any such vectors. The selection of vectorwill depend upon the preferred transformation technique and the targetspecies for transformation.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a plant cell host. Thesetechniques generally include transformation with DNA employing A.tumefaciens or A. rhizogenes as the transforming agent, liposomes, PEGprecipitation, electroporation, DNA injection, direct DNA uptake,microprojectile bombardment, particle acceleration, and the like (See,for example, EP 295959 and EP 138341) (see below). However, cells otherthan plant cells may be transformed with the expression cassettes of theinvention. The general descriptions of plant expression vectors andreporter genes, and Agrobacterium and Agrobacterium-mediated genetransfer, can be found in Gruber et al. (1993).

Expression vectors containing genomic or synthetic fragments can beintroduced into protoplasts or into intact tissues or isolated cells.Preferably expression vectors are introduced into intact tissue. Generalmethods of culturing plant tissues are provided for example by Maki etal., (1993); and by Phillips et al. (1988). Preferably, expressionvectors are introduced into maize or other plant tissues using a directgene transfer method such as microprojectile-mediated delivery, DNAinjection, electroporation and the like. More preferably expressionvectors are introduced into plant tissues using the microprojectilemedia delivery with the biolistic device. See, for example, Tomes et al.(1995). The vectors of the invention can not only be used for expressionof structural genes but may also be used in exon-trap cloning, orpromoter trap procedures to detect differential gene expression invarieties of tissues (Lindsey 1993; Auch & Reth 1990).

It is particularly preferred to use the binary type vectors of Ti and Riplasmids of Agrobacterium spp. Ti-derived vectors transform a widevariety of higher plants, including monocotyledonous and dicotyledonousplants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti1985: Byrne 1987; Sukhapinda 1987; Lorz 1985; Potrykus, 1985; Park 1985:Hiei 1994). The use of T-DNA to transform plant cells has receivedextensive study and is amply described (EP 120516; Hoekema, 1985; Knauf,1983; and An 1985). For introduction into plants, the chimeric genes ofthe invention can be inserted into binary vectors as described in theexamples.

Other transformation methods are available to those skilled in the art,such as direct uptake of foreign DNA constructs (see EP 295959),techniques of electroporation (Fromm 1986) or high velocity ballisticbombardment with metal particles coated with the nucleic acid constructs(Kline 1987, and U.S. Pat. No. 4,945,050). Once transformed, the cellscan be regenerated by those skilled in the art. Of particular relevanceare the recently described methods to transform foreign genes intocommercially important crops, such as rapeseed (De Block 1989),sunflower (Everett 1987), soybean (McCabe 1988; Hinchee 1988; Chee 1989;Christou 1989; EP 301749), rice (Hiei 1994), and corn (Gordon-Kamm 1990;Fromm 1990).

Those skilled in the art will appreciate that the choice of method mightdepend on the type of plant, i.e., monocotyledonous or dicotyledonous,targeted for transformation. Suitable methods of transforming plantcells include, but are not limited to, microinjection (Crossway 1986),electroporation (Riggs 1986), Agrobacterium-mediated transformation(Hinchee 1988), direct gene transfer (Paszkowski 1984), and ballisticparticle acceleration using devices available from Agracetus, Inc.,Madison, Wis. And BioRad, Hercules, Calif. (see, for example, U.S. Pat.No. 4,945,050; and McCabe 1988). Also see, Weissinger 1988; Sanford 1987(onion); Christou 1988 (soybean); McCabe 1988 (soybean); Datta 1990(rice); Klein 1988 (maize); Klein 1988 (maize); Klein 1988 (maize);Fromm 1990 (maize); and Gordon-Kamm 1990 (maize); Svab 1990 (tobaccochloroplast); Koziel 1993 (maize); Shimamoto 1989 (rice); Christou 1991(rice); European Patent Application EP 0 332 581 (orchardgrass and otherPooideae); Vasil 1993 (wheat); Weeks 1993 (wheat).

In another embodiment, a nucleotide sequence of the present invention isdirectly transformed into the plastid genome. Plastid transformationtechnology is extensively described in U.S. Pat. Nos. 5,451,513,5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and inMcBride et al., 1994. The basic technique for chloroplast transformationinvolves introducing regions of cloned plastid DNA flanking a selectablemarker together with the gene of interest into a suitable target tissue,e.g., using biolistics or protoplast transformation (e.g., calciumchloride or PEG mediated transformation). The 1 to 1.5 kb flankingregions, termed targeting sequences, facilitate orthologousrecombination with the plastid genome and thus allow the replacement ormodification of specific regions of the plastome. Initially, pointmutations in the chloroplast 16S rRNA and rps12 genes conferringresistance to spectinomycin and/or streptomycin are utilized asselectable markers for transformation (Svab 1990; Staub 1992). Thisresulted in stable homoplasmic transformants at a frequency ofapproximately one per 100 bombardments of target leaves. The presence ofcloning sites between these markers allowed creation of a plastidtargeting vector for introduction of foreign genes (Staub 1993).Substantial increases in transformation frequency are obtained byreplacement of the recessive rRNA or r-protein antibiotic resistancegenes with a dominant selectable marker, the bacterial aadA geneencoding the spectinomycin-detoxifying enzymeaminoglycoside-3N-adenyltransferase (Svab 1993). Other selectablemarkers useful for plastid transformation are known in the art andencompassed within the scope of the invention. Typically, approximately15-20 cell division cycles following transformation are required toreach a homoplastidic state. Plastid expression, in which genes areinserted by orthologous recombination into all of the several thousandcopies of the circular plastid genome present in each plant cell, takesadvantage of the enormous copy number advantage over nuclear-expressedgenes to permit expression levels that can readily exceed 10% of thetotal soluble plant protein. In a preferred embodiment, a nucleotidesequence of the present invention is inserted into a plastid targetingvector and transformed into the plastid genome of a desired plant host.Plants homoplastic for plastid genomes containing a nucleotide sequenceof the present invention are obtained, and are preferentially capable ofhigh expression of the nucleotide sequence.

Agrobacterium tumefaciens cells containing a vector comprising anexpression cassette of the present invention, wherein the vectorcomprises a Ti plasmid, are useful in methods of making transformedplants. Plant cells are infected with an Agrobacterium tumefaciens asdescribed above to produce a transformed plant cell, and then a plant isregenerated from the transformed plant cell. Numerous Agrobacteriumvector systems useful in carrying out the present invention are known.

Various Agrobacterium strains can be employed, preferably disarmedAgrobacterium tumefaciens or rhizogenes strains. In a preferredembodiment, Agrobacterium strains for use in the practice of theinvention include octopine strains, e.g., LBA4404 or agropine strains,e.g., EHA101 or EHA105. Suitable strains of A. tumefaciens for DNAtransfer are for example EHA101[pEHA101] (Hood 1986), EHA105[pEHA105](Li 1992), LBA4404[pAL4404] (Hoekema 1983), C58C1[pMP90] (Koncz & Schell1986), and C58C1[pGV2260] (Deblaere 1985). Other suitable strains areAgrobacterium tumefaciens C58, a nopaline strain. Other suitable strainsare A. tumefaciens C58C1 (Van Larebeke 1974), A136 (Watson 1975) orLBA4011 (Klapwijk 1980). In another preferred embodiment the soil-bornebacterium is a disarmed variant of Agrobacterium rhizogenes strain K599(NCPPB 2659). Preferably, these strains are comprising a disarmedplasmid variant of a Ti- or Ri-plasmid providing the functions requiredfor T-DNA transfer into plant cells (e.g., the vir genes). In apreferred embodiment, the Agrobacterium strain used to transform theplant tissue pre-cultured with the plant phenolic compound contains aL,L-succinamopine type Ti-plasmid, preferably disarmed, such as pEHA101.In another preferred embodiment, the Agrobacterium strain used totransform the plant tissue pre-cultured with the plant phenolic compoundcontains an octopine-type Ti-plasmid, preferably disarmed, such aspAL4404. Generally, when using octopine-type Ti-plasmids or helperplasmids, it is preferred that the virF gene be deleted or inactivated(Jarschow 1991).

The method of the invention can also be used in combination withparticular Agrobacterium strains, to further increase the transformationefficiency, such as Agrobacterium strains wherein the vir geneexpression and/or induction thereof is altered due to the presence ofmutant or chimeric virA or virG genes (e.g. Hansen 1994; Chen and Winans1991; Scheeren-Groot, 1994). Preferred are further combinations ofAgrobacterium tumefaciens strain LBA4404 (Hiei 1994) with super-virulentplasmids. These are preferably pTOK246-based vectors (Ishida 1996).

A binary vector or any other vector can be modified by common DNArecombination techniques, multiplied in E. coli, and introduced intoAgrobacterium by e.g., electroporation or other transformationtechniques (Mozo & Hooykaas 1991).

Agrobacterium is grown and used in a manner similar to that described inIshida (1996). The vector comprising Agrobacterium strain may, forexample, be grown for 3 days on YP medium (5 g/L yeast extract, 10 g/Lpeptone, 5 g/L NaCl, 15 g/L agar, pH 6.8) supplemented with theappropriate antibiotic (e.g., 50 mg/L spectinomycin). Bacteria arecollected with a loop from the solid medium and resuspended. In apreferred embodiment of the invention, Agrobacterium cultures arestarted by use of aliquots frozen at −80° C. The concentration ofAgrobacterium used for infection and co-cultivation may need to bevaried. For example, a cell suspension of the Agrobacterium having apopulation density of approximately from 10⁵ to 10¹¹, preferably 10⁶ to10¹⁰, more preferably about 10⁸ cells or cfu/mL is prepared and thetarget tissue is immersed in this suspension for about 3 to 10 minutes.The bacteria are resuspended in a plant compatible co-cultivationmedium. Supplementation of the co-culture medium with anti-oxidants(e.g., silver nitrate), phenol-absorbing compounds (likepolyvinylpyrrolidone, Perl 1996) or thiol compounds (e.g.,dithiothreitol, L-cysteine, Olhoft 2001) which can decrease tissuenecrosis due to plant defense responses (like phenolic oxidation) mayfurther improve the efficiency of Agrobacterium-mediated transformation.In another preferred embodiment, the co-cultivation medium of comprisesleast one thiol compound, preferably selected from the group consistingof sodium thiolsulfate, dithiotrietol (DTT) and cysteine. Preferably theconcentration is between about 1 mM and 10 mM of L-Cysteine, 0.1 mM to 5mM DTT, and/or 0.1 mM to 5 mM sodium thiolsulfate. Preferably, themedium employed during co-cultivation comprises from about 1 μM to about10 μM of silver nitrate and from about 50 mg/L to about 1,000 mg/L ofL-Cystein. This results in a highly reduced vulnerability of the targettissue against Agrobacterium-mediated damage (such as induced necrosis)and highly improves overall transformation efficiency.

Various vector systems can be used in combination with Agrobacteria.Preferred are binary vector systems. Common binary vectors are based on“broad host range”-plasmids like pRK252 (Bevan 1984) or pTJS75 (Watson1985) derived from the P-type plasmid RK2. Most of these vectors arederivatives of pBIN19 (Bevan 1984). Various binary vectors are known,some of which are commercially available such as, for example, pBI101.2or pBIN19 (Clontech Laboratories, Inc. USA). Additional vectors wereimproved with regard to size and handling (e.g. pPZP; Hajdukiewicz1994). Improved vector systems are described also in WO 02/00900.

Methods using either a form of direct gene transfer orAgrobacterium-mediated transfer usually, but not necessarily, areundertaken with a selectable marker which may provide resistance to anantibiotic (e.g., kanamycin, hygromycin or methotrexate) or a herbicide(e.g., phosphinothricin). The choice of selectable marker for planttransformation is not, however, critical to the invention.

For certain plant species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformation include the nptII gene which confers resistance tokanamycin and related antibiotics (Messing & Vierra, 1982; Bevan 1983),the bar gene which confers resistance to the herbicide phosphinothricin(White 1990, Spencer 1990), the hph gene which confers resistance to theantibiotic hygromycin (Blochlinger & Diggelmann), and the dhfr gene,which confers resistance to methotrexate (Bourouis 1983).

5. Production and Characterization of Stably Transformed Plants

Transgenic plant cells are then placed in an appropriate selectivemedium for selection of transgenic cells which are then grown to callus.Shoots are grown from callus and plantlets generated from the shoot bygrowing in rooting medium. The various constructs normally will bejoined to a marker for selection in plant cells. Conveniently, themarker may be resistance to a biocide (particularly an antibiotic, suchas kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide,or the like). The particular marker used will allow for selection oftransformed cells as compared to cells lacking the DNA which has beenintroduced. Components of DNA constructs including transcriptioncassettes of this invention may be prepared from sequences, which arenative (endogenous) or foreign (exogenous) to the host. By “foreign” itis meant that the sequence is not found in the wild-type host into whichthe construct is introduced. Heterologous constructs will contain atleast one region which is not native to the gene from which thetranscription-initiation-region is derived.

To confirm the presence of the transgenes in transgenic cells andplants, a variety of assays may be performed. Such assays include, forexample, “molecular biological” assays well known to those of skill inthe art, such as Southern and Northern blotting, in situ hybridizationand nucleic acid-based amplification methods such as PCR or RT-PCR;“biochemical” assays, such as detecting the presence of a proteinproduct, e.g., by immunological means (ELISAs and Western blots) or byenzymatic function; plant part assays, such as seed assays; and also, byanalyzing the phenotype of the whole regenerated plant, e.g., fordisease or pest resistance.

DNA may be isolated from cell lines or any plant parts to determine thepresence of the preselected nucleic acid segment through the use oftechniques well known to those skilled in the art. Note that intactsequences will not always be present, presumably due to rearrangement ordeletion of sequences in the cell.

The presence of nucleic acid elements introduced through the methods ofthis invention may be determined by polymerase chain reaction (PCR).Using this technique discreet fragments of nucleic acid are amplifiedand detected by gel electrophoresis. This type of analysis permits oneto determine whether a preselected nucleic acid segment is present in astable transformant, but does not prove integration of the introducedpreselected nucleic acid segment into the host cell genome. In addition,it is not possible using PCR techniques to determine whethertransformants have exogenous genes introduced into different sites inthe, genome, i.e., whether transformants are of independent origin. Itis contemplated that using PCR techniques it would be possible to clonefragments of the host genomic DNA adjacent to an introduced preselectedDNA segment.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced preselected DNAsegments in high molecular weight DNA, i.e., confirm that the introducedpreselected, DNA segment has been integrated into the host cell genome.The technique of Southern hybridization provides information that isobtained using PCR, e.g., the presence of a preselected DNA segment, butalso demonstrates integration into the genome and characterizes eachindividual transformant.

It is contemplated that using the techniques of dot or slot blothybridization which are modifications of Southern hybridizationtechniques one could obtain the same information that is derived fromPCR, e.g., the presence of a preselected DNA segment. Both PCR andSouthern hybridization techniques can be used to demonstratetransmission of a preselected DNA segment to progeny. In most instancesthe characteristic Southern hybridization pattern for a giventransformant will segregate in progeny as one or more Mendelian genes(Spencer 1992); Laursen 1994) indicating stable inheritance of the gene.The non-chimeric nature of the callus and the parental transformants(R.sub.0) was suggested by germline transmission and the identicalSouthern blot hybridization patterns and intensities of the transformingDNA in callus, R.sub.0 plants and R.sub.1 progeny that segregated forthe transformed gene.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA may only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR techniques may also be used for detection andquantification of RNA produced from introduced preselected DNA segments.In this application of PCR it is first necessary to reverse transcribeRNA into DNA, using enzymes such as reverse transcriptase, and thenthrough the use of conventional PCR techniques amplify the DNA. In mostinstances PCR techniques, while useful, will not demonstrate integrityof the RNA product. Further information about the nature of the RNAproduct may be obtained by Northern blotting. This technique willdemonstrate the presence of an RNA species and give information aboutthe integrity of that RNA. The presence or absence of an RNA species canalso be determined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and will onlydemonstrate the presence or absence of an RNA species.

While Southern blotting and PCR may be used to detect the preselectedDNA segment in question, they do not provide information as to whetherthe preselected DNA segment is being expressed. Expression may beevaluated by specifically identifying the protein products of theintroduced preselected DNA segments or evaluating the phenotypic changesbrought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as Western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Assay procedures may also be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to beanalyzed.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Morphological changes may include greater stature or thickerstalks. Most often changes in response of plants or plant parts toimposed treatments are evaluated under carefully controlled conditionstermed bioassays.

6. Uses of Transgenic Plants

Once an expression cassette of the invention has been transformed into aparticular plant species, it may be propagated in that species or movedinto other varieties of the same species, particularly includingcommercial varieties, using traditional breeding techniques.Particularly preferred plants of the invention include the agronomicallyimportant crops listed above. The genetic properties engineered into thetransgenic seeds and plants described above are passed on by sexualreproduction and can thus be maintained and propagated in progenyplants. The present invention also relates to a transgenic plant cell,tissue, organ, seed or plant part obtained from the transgenic plant.Also included within the invention are transgenic descendants of theplant as well as transgenic plant cells, tissues, organs, seeds andplant parts obtained from the descendants.

Preferably, the expression cassette in the transgenic plant is sexuallytransmitted. In one preferred embodiment, the coding sequence issexually transmitted through a complete normal sexual cycle of the R0plant to the R1 generation. Additionally preferred, the expressioncassette is expressed in the cells, tissues, seeds or plant of atransgenic plant in an amount that is different than the amount in thecells, tissues, seeds or plant of a plant which only differs in that theexpression cassette is absent.

The transgenic plants produced herein are thus expected to be useful fora variety of commercial and research purposes. Transgenic plants can becreated for use in traditional agriculture to possess traits beneficialto the grower (e.g., agronomic traits such as resistance to waterdeficit, pest resistance, herbicide resistance or increased yield),beneficial to the consumer of the grain harvested from the plant (e.g.,improved nutritive content in human food or animal feed; increasedvitamin, amino acid, and antioxidant content; the production ofantibodies (passive immunization) and nutriceuticals), or beneficial tothe food processor (e.g., improved processing traits). In such uses, theplants are generally grown for the use of their grain in human or animalfoods. Additionally, the use of root-specific promoters in transgenicplants can provide beneficial traits that are localized in theconsumable (by animals and humans) roots of plants such as carrots,parsnips, and beets. However, other parts of the plants, includingstalks, husks, vegetative parts, and the like, may also have utility,including use as part of animal silage or for ornamental purposes.Often, chemical constituents (e.g., oils or starches) of maize and othercrops are extracted for foods or industrial use and transgenic plantsmay be created which have enhanced or modified levels of suchcomponents.

Transgenic plants may also find use in the commercial manufacture ofproteins or other molecules, where the molecule of interest is extractedor purified from plant parts, seeds, and the like. Cells or tissue fromthe plants may also be cultured, grown in vitro, or fermented tomanufacture such molecules. The transgenic plants may also be used incommercial breeding programs, or may be crossed or bred to plants ofrelated crop species. Improvements encoded by the expression cassettemay be transferred, e.g., from maize cells to cells of other species,e.g., by protoplast fusion.

The transgenic plants may have many uses in research or breeding,including creation of new mutant plants through insertional mutagenesis,in order to identify beneficial mutants that might later be created bytraditional mutation and selection. An example would be the introductionof a recombinant DNA sequence encoding a transposable element that maybe used for generating genetic variation. The methods of the inventionmay also be used to create plants having unique “signature sequences” orother marker sequences which can be used to identify proprietary linesor varieties.

Thus, the transgenic plants and seeds according to the invention can beused in plant breeding, which aims at the development of plants withimproved properties conferred by the expression cassette, such astolerance of drought, disease, or other stresses. The various breedingsteps are characterized by well-defined human intervention such asselecting the lines to be crossed, directing pollination of the parentallines, or selecting appropriate descendant plants. Depending on thedesired properties different breeding measures are taken. The relevanttechniques are well known in the art and include but are not limited tohybridization, inbreeding, backcross breeding, multilane breeding,variety blend, interspecific hybridization, aneuploid techniques, etc.Hybridization techniques also include the sterilization of plants toyield male or female sterile plants by mechanical, chemical orbiochemical means. Cross pollination of a male sterile plant with pollenof a different line assures that the genome of the male sterile butfemale fertile plant will uniformly obtain properties of both parentallines. Thus, the transgenic seeds and plants according to the inventioncan be used for the breeding of improved plant lines which for exampleincrease the effectiveness of conventional methods such as herbicide orpesticide treatment or allow to dispense with said methods due to theirmodified genetic properties. Alternatively new crops with improvedstress tolerance can be obtained which, due to their optimized genetic“equipment”, yield harvested product of better quality than products,which were not able to tolerate comparable adverse developmentalconditions.

Sequences

-   1. SEQ ID NO: 1 Nucleic acid sequence encoding the transcription    regulating nucleotide sequence of Oryza sativa (rice) caffeoyl    CoA-O-methyltransferase (Os.CCoAMT1) gene including 5′-untranslated    region-   2. SEQ ID NO: 2 Nucleic acid sequence encoding the transcription    regulating nucleotide sequence of Oryza sativa (rice) caffeoyl    CoA-O-methyltransferase (Os.CCoAMT1) gene-   3. SEQ ID NO: 3 Nucleic acid sequence encoding the core promoter    region of the transcription regulating nucleotide sequence of Oryza    sativa (rice) caffeoyl CoA-O-methyltransferase (Os.CCoAMT1) gene    comprising clusters of promoter elements.-   4. SEQ ID NO: 4 Nucleic acid sequence encoding Oryza sativa (rice)    caffeoyl CoA-O-methyltransferase (Os.CCoAMT1)-   5. SEQ ID NO: 5 Amino acid sequence encoding Oryza sativa (rice)    caffeoyl CoA-O-methyltransferase (Os.CCoAMT1)-   6. SEQ ID NO: 6 Nucleic acid sequence encoding a transcription    regulating nucleotide sequence from the Oryza sativa (rice)    C8,7-sterol isomerase gene (Os.SI) including the 5′ untranslated    region of the gene.-   7. SEQ ID NO: 7 Nucleic acid sequence encoding a transcription    regulating nucleotide sequence from the Oryza sativa (rice)    C8,7-sterol isomerase gene (Os.SI).-   8. SEQ ID NO: 8 Nucleic acid sequence encoding the core promoter    region of the transcription regulating nucleotide sequence from the    Oryza sativa (rice) C8,7-sterol isomerase gene (Os.SI) comprising    clusters of promoter elements.-   9. SEQ ID NO: 9 Nucleic acid sequence encoding Oryza sativa (rice)    C8,7-sterol isomerase gene (Os.SI)-   10. SEQ ID NO: 10 Amino acid sequence encoding Oryza sativa (rice)    C8,7-sterol isomerase gene (Os.SI)-   11. SEQ ID NO: 11 Nucleic acid sequence encoding a transcription    regulating nucleotide sequence from a Zea mays hydroxyproline-rich    glycoprotein (HRGP) (Zm.HRGP) including the 5′ untranslated region    of the gene.-   12. SEQ ID NO: 12 Nucleic acid sequence encoding a transcription    regulating nucleotide sequence from a Zea mays hydroxyproline-rich    glycoprotein (HRGP) (Zm.HRGP).-   13. SEQ ID NO: 13 Nucleic acid sequence encoding the core promoter    region of the transcription regulating nucleotide sequence from a    Zea mays hydroxyproline-rich glycoprotein (HRGP) (Zm.HRGP)    comprising clusters of promoter elements.-   14. SEQ ID NO: 14 Nucleic acid sequence encoding a function    equivalent of the transcription regulating nucleotide sequence from    a Zea mays hydroxyproline-rich glycoprotein (HRGP) (Zm.HRGP)    including the 5′ untranslated region of the gene.-   15. SEQ ID NO: 15 Nucleic acid sequence encoding a function    equivalent of the transcription regulating nucleotide sequence from    a Zea mays hydroxyproline-rich glycoprotein (HRGP) (Zm.HRGP).-   16. SEQ ID NO: 16 Nucleic acid sequence encoding the core promoter    region of a function equivalent of the transcription regulating    nucleotide sequence from a Zea mays hydroxyproline-rich glycoprotein    (HRGP) (Zm.HRGP) comprising clusters of promoter elements.-   17. SEQ ID NO: 17 Nucleic acid sequence encoding the Zea mays    hydroxyproline-rich glycoprotein (HRGP)-   18. SEQ ID NO: 18 Amino acid sequence encoding the Zea mays    hydroxyproline-rich glycoprotein (HRGP)-   19. SEQ ID NO: 19 Nucleic acid sequence encoding a transcription    regulating nucleotide sequence from a Zea mays lactate dehydrogenase    (Zm.LDH) including the 5′ untranslated region of the gene.-   20. SEQ ID NO: 20 Nucleic acid sequence encoding a transcription    regulating nucleotide sequence from a Zea mays lactate dehydrogenase    (Zm.LDH).-   21. SEQ ID NO: 21 Nucleic acid sequence encoding the core promoter    region of the transcription regulating nucleotide sequence from a    Zea mays lactate dehydrogenase (Zm.LDH) comprising clusters of    promoter elements.-   22. SEQ ID NO: 22 Nucleic acid sequence encoding a function    equivalent of the transcription regulating nucleotide sequence from    a Zea mays lactate dehydrogenase (Zm.LDH) including the 5′    untranslated region of the gene.-   23. SEQ ID NO: 23 Nucleic acid sequence encoding a function    equivalent of the transcription regulating nucleotide sequence from    a Zea mays lactate dehydrogenase (Zm.LDH).-   24. SEQ ID NO: 24 Nucleic acid sequence encoding the core promoter    region of a function equivalent of the transcription regulating    nucleotide sequence from a Zea mays lactate dehydrogenase (Zm.LDH)    comprising clusters of promoter elements.-   25. SEQ ID NO: 25 Nucleic acid sequence encoding the Zea mays    lactate dehydrogenase (Zm.LDH)-   26. SEQ ID NO: 26 Amino acid sequence encoding the Zea mays lactate    dehydrogenase (Zm.LDH)-   27. SEQ ID NO: 27 Nucleic acid sequence encoding a function    equivalent of the transcription regulating nucleotide sequence from    an Oryza sativa (rice) choroplast 12 (CP12) protein including the 5′    untranslated region of the gene.-   28. SEQ ID NO: 28 Nucleic acid sequence encoding a function    equivalent of the transcription regulating nucleotide sequence from    an Oryza sativa (rice) choroplast 12 (CP12) protein.-   29. SEQ ID NO: 29 Nucleic acid sequence encoding the core promoter    region of a function equivalent of the transcription regulating    nucleotide sequence from an Oryza sativa (rice) choroplast 12 (CP12)    protein comprising clusters of promoter elements.-   30. SEQ ID NO: 30 Nucleic acid sequence encoding the Oryza sativa    (rice) choroplast 12 (CP12) protein.-   31. SEQ ID NO: 31 Amino acid sequence encoding the Oryza sativa    (rice) choroplast 12 (CP12) protein.-   32. SEQ ID NO: 32 Nucleic acid sequence encoding the intergenic    sequence comprising 3′-untranslated region of Zea mays lactate    dehydrogenase gene with the transcription termination and    polyadenylation sequence.-   33. SEQ ID NO: 33 Nucleic acid sequence encoding construct pBPSMM304    [Os.CP12 promoter::Zm.ubiquitin intron::GUS (PIV2)::NOS terminator]-   34. SEQ ID NO: 34 Nucleic acid sequence encoding the intergenic    sequence including the 3′ untranslated region of caffeoyl    CoA-O-methyltransferase with the transcription termination and    polyadenylation sequence.-   35. SEQ ID NO: 35 Nucleic acid sequence encoding the intergenic    sequence including the 3′ untranslated region of hydroxyproline-rich    glyco-protein gene with the transcription termination and    polyadenylation sequence.-   36. SEQ ID NO: 36 Nucleic acid sequence encoding construct pBPS325    [Os.CCoAMT1 promoter::Zm.ubiquitin intron::GUS (PIV2)::Os.CCoAMT1    terminator]-   37. SEQ ID NO: 37 Nucleic acid sequence encoding construct pBPS331    [Os.SI promoter::Zm.ubiquitin intron::GUS (PIV2)::NOS terminator]-   38. SEQ ID NO: 38 Nucleic acid sequence encoding construct pBPSET003    [Zm.HRGP promoter::Zm.ubiquitin intron::GUS (PIV2)::Zm.HRGP    terminator]-   39. SEQ ID NO: 39 Nucleic acid sequence encoding construct pBPSET007    [Zm.LDH promoter::Zm.ubiquitin intron::GUS (PIV2)::Zm.LDH    terminator]

40. Forward Primer Os.CCoAMT1 promoter-5′ SEQ ID NO: 405′-CAACTACTGCACGGTAAAAGTGATAGG-3′41. Reverse primer Os.CCoAMT1 promoter-3′ SEQ ID NO: 415′-GCAGCTTGCTTCGATCTCTCGCTCGCC-3′ 42. Forward Primer Os.CCoAMT1 3′UTR-5′SEQ ID NO: 42 5′-GCCGATGCCCAAGAACTAGTCATTTTAA-3′43 Reverse primer Os.CCoAMT1 3′UTR-3′ SEQ ID NO: 435′-ATTAACACGTCAACCAAACCGCCGTCC-3′ 44 Forward Primer Os.SI promoter-5′SEQ ID NO: 44 5′-TGCCTCGATTCGACCGTGTAATGGAAT-3′45. Reverse primer Os.SI promoter-3′ SEQ ID NO: 455′-ACTCCTGGCTTCCTTCCGATCTGGACT-3′ 46. Forward Primer Zm.HRGP promoter-5′SEQ ID NO: 46 5′-CCGGTGACCTTCTTGCTTCTTCGATCG-3′47. Reverse primer Zm.HRGP promoter-3′ SEQ ID NO: 475′-CCTCTCTCTCACACACACTCTCAGTAA-3′ 48. Forward primer ZmLDH promoter-5′SEQ ID NO: 48 5′-AACAAATGGCGTACTTATATAACCACA-3′49. Reverse primer ZmLDH promoter-3′ SEQ ID NO: 495′-CGGGCGGAATGGGATGGGATTACGTGT-3′ 50. Forward primer Zm.HRGP 3′UTR-5′SEQ ID NO: 50 5′-AAAGCGATGCCTACCATACCACACTGC-3′51. Reverse primer Zm.HRGP 3′UTR-3′ SEQ ID NO: 515′-TGCCCACATTTATTATGGTTTTACACCC-3′ 52. Forward Primer Zm.LDH 3′UTR-5′SEQ ID NO: 52 5′-TGATCACATCACCGTCTCTCTTCATTAA-3′53. Reverse primer Zm.LDH 3′UTR-3′ SEQ ID NO: 535′-TATCCCAGTCTCGATATTGTCATCCGCT-3′ 54. Forward primer Os.CP12-p FPSEQ ID NO: 54 5′-TTTGTATTTAGGTCCCTAACGCCCTC-3′55. Reverse primer Os.CP12-p RP SEQ ID NO: 555′-TGTTGATGCGGATTTCTGCGTGTGAT-3′

-   56. SEQ ID NO: 56 Nucleic acid sequence encoding a transcription    regulating nucleotide sequence from a Oryza sativa lactate    dehydrogenase (Os.LDH) including the 5′ untranslated region of the    gene.-   57. SEQ ID NO: 57 Nucleic acid sequence encoding a transcription    regulating nucleotide sequence from a Oryza sativa lactate    dehydrogenase (Os.LDH).-   58. SEQ ID NO: 58 Nucleic acid sequence encoding the core promoter    region of the transcription regulating nucleotide sequence from a    Oryza sativa lactate dehydrogenase (Os.LDH) comprising clusters of    promoter elements.-   59. SEQ ID NO: 59 Nucleic acid sequence encoding a Oryza sativa    lactate dehydrogenase (Os.LDH)-   60. SEQ ID NO: 61 Amino acid sequence encoding a Oryza sativa    lactate dehydrogenase (Os.LDH)-   61. SEQ ID NO: 61 Nucleic acid sequence encoding a transcription    regulating nucleotide sequence from a Oryza sativa lactate    dehydrogenase (Os.LDH) including the 5′ untranslated region of the    gene.-   62. SEQ ID NO: 62 Nucleic acid sequence encoding a transcription    regulating nucleotide sequence from a Oryza sativa lactate    dehydrogenase (Os.LDH).-   63. SEQ ID NO: 63 Nucleic acid sequence encoding the core promoter    region of the transcription regulating nucleotide sequence from a    Oryza sativa lactate dehydrogenase (Os.LDH) comprising clusters of    promoter elements.-   64. SEQ ID NO: 64 Nucleic acid sequence encoding a Oryza sativa    lactate dehydrogenase (Os.LDH)-   65. SEQ ID NO: 65 Amino acid sequence encoding a Oryza sativa    lactate dehydrogenase (Os.LDH)-   66. SEQ ID NO: 66 Nucleic acid sequence encoding the transcription    regulating nucleotide sequence of Zea mays caffeoyl    CoA-O-methyltransferase (Zm.CCoAMT1) gene including 5′-untranslated    region-   67. SEQ ID NO: 67 Nucleic acid sequence encoding the transcription    regulating nucleotide sequence of Zea mays caffeoyl    CoA-O-methyltransferase (Zm.CCoAMT1) gene-   68. SEQ ID NO: 68 Nucleic acid sequence encoding the core promoter    region of the transcription regulating nucleotide sequence of Zea    mays caffeoyl CoA-O-methyltransferase (Zm.CCoAMT1) gene comprising    clusters of promoter elements.-   69. SEQ ID NO: 69 Nucleic acid sequence encoding Zea mays caffeoyl    CoA-O-methyltransferase (Zm.CCoAMT1)-   70. SEQ ID NO: 70 Amino acid sequence encoding Zea mays caffeoyl    CoA-O-methyltransferase (Zm.CCoAMT1)-   71. SEQ ID NO: 71 Nucleic acid sequence encoding a function    equivalent of the transcription regulating nucleotide sequence from    a Zea diploperennis hydroxyproline-rich glycoprotein (HRGP)    including the 5′ untranslated region of the gene.-   72. SEQ ID NO: 72 Nucleic acid sequence encoding a function    equivalent of the transcription regulating nucleotide sequence from    a Zea diploperennis hydroxyproline-rich glycoprotein (HRGP).-   73. SEQ ID NO: 73 Nucleic acid sequence encoding the core promoter    region of a function equivalent of the transcription regulating    nucleotide sequence from a Zea diploperennis hydroxyproline-rich    glycoprotein (HRGP) comprising clusters of promoter elements.-   74. SEQ ID NO: 74 Nucleic acid sequence encoding the Zea    diploperennis hydroxyproline-rich glycoprotein (HRGP)-   75. SEQ ID NO: 75 Amino acid sequence encoding the Zea diploperennis    hydroxyproline-rich glycoprotein (HRGP)-   76. SEQ ID NO: 76-84 Amino acid sequence motif of a monocotyledonous    plant lactate dehydrogenase protein-   77. SEQ ID NO: 85-90 Amino acid sequence motif of a monocotyledonous    plant caffeoyl-CaA-O-methyltransferase protein

78. Oligonucleotide primer GUS-forward: SEQ ID NO: 915′-ttacgtggcaaaggattcgat-3′ 79. Oligonucleotide primer GUS-reverse:SEQ ID NO: 92 5′-gccccaatccagtccattaa-380. Oligonucleotide primer Control gene forward SEQ ID NO: 935′-tctgccttgcccttgctt-3′ 81. Oligonucleotide primer Control gene reverseSEQ ID NO: 94 5′-caattgcttggcaggtatattt-3′

EXAMPLES Materials and General Methods

Unless indicated otherwise, chemicals and reagents in the Examples wereobtained from Sigma Chemical Company (St. Louis, Mo.), restrictionendonucleases were from New England Biolabs (Beverly, Mass.) or Roche(Indianapolis, Ind.), oligonucleotides were synthesized by MWG BiotechInc. (High Point, N.C.), and other modifying enzymes or kits regardingbiochemicals and molecular biological assays were from Clontech (PaloAlto, Calif.), Pharmacia Biotech (Piscataway, N.J.), Promega Corporation(Madison, Wis.), or Stratagene (La Jolla, Calif.). Materials for cellculture media were obtained from Gibco/BRL (Gaithersburg, Md.) or DIFCO(Detroit, Mich.). The cloning steps carried out for the purposes of thepresent invention, such as, for example, restriction cleavages, agarosegel electrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linking DNA fragments,transformation of E. coli cells, growing bacteria, multiplying phagesand sequence analysis of recombinant DNA, are carried out as describedby Sambrook (1989). The sequencing of recombinant DNA molecules iscarried out using ABI laser fluorescence DNA sequencer following themethod of Sanger (Sanger 1977).

For generating transgenic plants Agrobacterium tumefaciens (strainC58C1[pMP90]) is transformed with the various promoter::GUS vectorconstructs (see below). Resulting Agrobacterium strains are subsequentlyemployed to obtain transgenic plants. For this purpose a isolatedtransformed Agrobacterium colony is incubated in 4 mL culture (Medium:YEB medium with 50 μg/mL Kanamycin and 25 μg/mL Rifampicin) over nightat 28° C. With this culture a 400 ml culture of the same medium isinoculated and incubated over night (28° C., 220 rpm). The bacteria aprecipitated by centrifugation (GSA-Rotor, 8,000 U/min, 20 min) and thepellet is resuspended in infiltration medium (½ MS-Medium; 0.5 g/L MES,pH 5.8; 50 g/L sucrose). The suspension is placed in a plant box(Duchefa) and 100 mL SILVET L-77 (Osi Special-ties Inc., Cat. P030196)are added to a final concentration of 0.02%. The plant box with 8 to 12Plants is placed into a desiccator for 10 to 15 min. under vacuum withsubsequent, spontaneous ventilation (expansion). This process isrepeated 2-3 times. Thereafter all plants are transferred into pods withwet-soil and grown under long daytime conditions (16 h light; daytemperature 22-24° C., night temperature 19° C.; 65% rel. humidity).Seeds are harvested after 6 weeks.

Example 1 Isolation of Promoters

Genomic DNA from maize and rice is extracted using the Qiagen DNAeasyPlant Mini Kit (Qiagen). The promoter regions were isolated from genomicDNA using conventional PCR. Approximately 0.1 μg of digested genomic DNAwas uses for the regular PCR reaction (see below). The primers weredesigned based on the maize or rice genomic DNA sequences upstream ofthe EST candidates, maize genomic sequences, or promoter sequencesdisclosed in the public database (e.g. rice caffeoylCoA-O-methyltransferase [CCoAMT1], GenBank accession number AB023482;rice unknown protein, GenBank accession number AP002818; maizehydroxyproline-rich glycoprotein [HRGP], GenBank accession numberAJ131535; maize lactate dehydrogenase [LDH], GenBank accession numberZ11754). One μL of the diluted digested genomic DNA was used as the DNAtemplate in the primary PCR reaction. The reaction comprised forward(5′) and reverse (3′) primers in a mixture containing Buffer 3 followingthe protocol outlined by an Expand Long PCR kit (Cat #1681-842,Roche-Boehringer Mannheim). The isolated DNA is employed as template DNAin a PCR amplification reaction using the following primers:

TABLE 3 Primer sequences for isolation of the promoter or terminator regionPromoter or Size Primer Sequences Terminator* (bp)Forward Primer (F) & Reverse Primer (R) Oryza sativa 1,035F: 5′-CAACTACTGCACGGTAAAAGTGATAGG-3′ Caffeoyl-CoA-O- (SEQ ID NO: 40)methyltransferase R: 5′-GCAGCTTGCTTCGATCTCTCGCTCGCC-3′ Promoter(SEQ ID NO: 41) (Os.CCoAMT1-p) Oryza sativa 1,092FP: 5′-GCCGATGCCCAAGAACTAGTCATTTTA-3′ Caffeoyl-CoA-O- (SEQ ID NO: 42)methyltransferase RP: 5′-ATTAACACGTCAACCAAACCGCCGTCC-3′ Terminator(SEQ ID NO: 43) (Os.CCoAMT1-t) Oryza sativa 813FP: 5′-TGCCTCGATTCGACCGTGTAATGGAAT-3′ C-8,7-sterol (SEQ ID NO: 44)isomerase RP: 5′-ACTCCTGGCTTCCTTCCGATCTGGACT-3′ Promoter (SEQ ID NO: 45)(Os.SI-p) Zea maize 1,263 FP: 5′-CCGGTGACCTTCTTGCTTCTTCGATCG-3′Hydroxyproline-rich (SEQ ID NO: 46) glycoprotein RP: 5′-CCTCTCTCTCACACACACTCTCAGTAA-3′ Promoter (SEQ ID NO: 47)(Zm.HRGP-p) Zea maize 541 FP: 5′-AAAGCGATGCCTACCATACCACACTGC-3′Hydroxyproline-rich (SEQ ID NO: 50) glycoprotein RP: 5′-TGCCCACATTTATTATGGTTTTACACCC-3′ Terminator (SEQ ID NO: 51)(Zm.HRGP-t) Zea maize 1,061 FP: 5′-AACAAATGGCGTACTTATATAACCACA-3′Lactate- (SEQ ID NO: 48) dehydrogenaseRP: 5′-CGGGCGGAATGGGATGGGATTACGTGT-3′ promoter (SEQ ID NO: 49)(Zm.LDH-p) Zea maize 475 FP: 5′-TGATCACATCACCGTCTCTCTTCATTAA-3′ Lactate-(SEQ ID NO: 52) dehydrogenase RP: 5′-TATCCCAGTCTCGATATTGTCATCCGCT-3′terminator (SEQ ID NO: 53) (Zm.LDH-t) Oryza sativa 998FP: 5′-TTTGTATTTAGGTCCCTAACGCCCTC-3′ Chloroplast protein (SEQ ID NO: 54)12 Promoter RP: 5′-TGTTGATGCGGATTTCTGCGTGTGAT-3′ (Os.CP12-p)(SEQ ID NO: 55) terminator including 3′UTR

The promoter regions are amplified in the reaction solution [1×PCRreaction buffer (Roche Diagnostics), 5 μL genomic DNA (corresponds toapproximately 80 ng, 2.5 mM of each dATP, dCTP, dGTP and dTTP(Invitrogen: dNTP mix), 1 μL 5′ primer (100 μM) 1 μL 3′ primer (100 μM),1 μL Taq DNA polymerase 5 U/4 (Roche Diagnostics), in a final volume of100 μL under the optimized PCR thermocycler program (T3 ThermocyclerBiometra; 1 cycle with 180 sec at 95° C., 30 cycles with 40 sec at 95°C., 60 sec at 53° C. and 2 min at 72° C., and 1 cycle with 5 min at 72°C. before stop the reaction at 4° C.).

The PCR product was applied to a 1% (w/v) agarose gel and separated at80V followed by excising from the gel and purified with the aid of theQiagen Gel Extraction Kit (Qiagen, Hilden, Germany). If appropriate, theeluate of 50 μL can be evaporated. The PCR product was cloned directlyinto vector pCR4-TOPO (Invitrogen) following the manufacturer'sinstructions, i.e. the PCR product obtained is inserted into a vectorhaving T overhangs with its A overhangs and a topoisomerase.

Example 2 Isolation of Terminator of Interest Including the 3′Untranslated Region

Rice genomic DNA fragment (1,092 bp) containing the 3′ untranslatedregion of caffeoyl CoA-O-methyltransferase (Os.CCoAMT1) was isolatedusing sequence specific primers based on the sequences that disclosed inthe public database (GenBank accession number AB023482). The protocolsfor plant genomic DNA isolation and conventional PCR amplification wasdescribed in the Example 1.

Forward Primer OsCCoAMT1 3′UTR-5′: (SEQ ID NO: 42)5′-GCCGATGCCCAAGAACTAGTCATTTTA-3′ Reverse primer OsCCoAMT1 3′UTR-3′:(SEQ ID NO: 43) 5′-ATTAACACGTCAACCAAACCGCCGTCC-3′

SacI for the forward primer and PmeI for the reverse primer were addedto the sequence-specific primers for the further cloning purpose. (Theillustrated primer sequences do not include restriction enzyme sites.)

Rice genomic DNA fragment, 519 bp or 473 bp, containing the 3′untranslated region of HRGP or LDH gene was isolated, respectively usingsequence specific primers based on the sequences that disclosed in thepublic database (GenBank accession number AJ131535; Z11754). Theprotocols for plant genomic DNA isolation and conventional PCRamplification using sequence specific primers was described in theExample 1.

SacI for the forward primer and PmeI for the reverse primer were addedto the sequence-specific primers for the further cloning purpose. (Theillustrated primer sequences do not include restriction enzyme sites.)

Example 3 Vector Construction 3.1 pUC Based Vector (Promoter ofInterest::Intron (IME)::GUS::NOS or Terminator of Interest)

The base vector to which the intron candidates were cloned in waspBPSMM270 at BglI and XmaI. This vector comprises multiple cloning sites(MCS) followed by Zm.ubiquitin intron, the GUSint ORF (including thepotato invertase [PIV]2 intron to prevent bacterial expression), andnopaline synthase (NOS) terminator in order (5′ to 3′). Maize ubiquitinintron can be replaced with an intron of interest that functions inintron-mediated enhancement.

The PCR fragment containing terminator of interest (e.g. 1,092 bp ricegenomic DNA including CCoAMT1 terminator; 558 bp maize genomic DNAincluding HRGP terminator, 477 bp maize genomic DNA including LDHterminator) was digested with SacI and PmeI enzymes. Nopaline synthaseterminator region in pBPSMM270 was replaced with the CCoAMT1 terminator,HRGP terminator or LDH terminator resulting in pBPSMM270a,pBPSMM270-HRGP3′ or pBPSMM270-LDH3′, respectively.

The genomic DNA fragment containing Os.CCoAMT1 or Os.SI promoter in theTopo vector (Invitrogen) was digested with PacI and AscI followed bysubcloned upstream of the Zm.ubiquitin intron in pBPSMM270,respectively.

The genomic DNA fragment containing CCoAMT1 promoter in the Topo vector(Invitrogen) was digested with PacI and AscI followed by subclonedupstream of the Zm.ubiquitin intron in pBPSMM270-CCoAMT1 3′,respectively.

The genomic DNA fragment containing Zm.HRGP or Zm.LDH promoter in theTopo vector (Invitrogen) was digested with PacI and AscI followed bysubcloned upstream of the Zm.ubiquitin intron in pBPSMM270-HRGP3′ orpBPSMM270-LDH3′, respectively.

3.2 Transformation Binary Vector (Promoter of Interest::Intron(IME)::GUS::NOS or Terminator of Interest)

The GUS chimeric cassette (Os.CCoAMT1 promoter::Zm.ubiquitin intron::GUS(PIV2)::CCoAMT1 terminator, Zm.CCoAMT1 promoter::Zm.ubiquitinintron::GUS (PIV2)::NOS, Os.SI promoter::Zm.ubiquitin intron::GUS(PIV2)::NOS, Zm.HRGP promoter::Zm.ubiquitin intron::GUS (PIV2)::Zm.HRGPterminator, or Zm.LDH promoter::Zm.ubiquitin intron::GUS (PIV2)::Zm.LDHterminator) in pUC-based vector were digested with AscI or PacI (5′) andPmeI (3′) and subcloned into a monocot binary vector containing a plantselectable marker cassette (pBPSMM344) at AscI or PacI (5′) and PmlI(3′) sites to generate pBPSMM325, pBPSMM271, pBPSMM331, pBPSET003, orpBPSET007, respectively.

Example 4 Agrobacterium-Mediated Transformation in MonocotyledonousPlants

The Agrobacterium-mediated plant transformation using standardtransformation and regeneration techniques may also be carried out forthe purposes of transforming crop plants (Gelvin 1995; Glick 1993).

The transformation of maize or other monocotyledonous plants can becarried out using, for example, a technique described in U.S. Pat. No.5,591,616.

The transformation of plants using particle bombardment, polyethyleneglycol-mediated DNA uptake or via the silicon carbonate fiber techniqueis described, for example, by Freeling & Walbot (1993) “The maizehandbook” ISBN 3-540-97826-7, Springer Verlag New York).

Example 5 Detection of Reporter Gene Expression

To identify the characteristics of the promoter and the essentialelements of the latter, which bring about its tissue specificity, it isnecessary to place the promoter itself and various fragments thereofbefore what is known as a reporter gene, which allows the determinationof the expression activity. An example, which may be mentioned, is thebacterial β-glucuronidase (Jefferson 1987a). The β-glucuronidaseactivity can be detected in-planta by means of a chromogenic substratesuch as 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid in an activitystaining (Jefferson 1987b). To study the tissue specificity, the planttissue is cut, embedded, stained and analyzed as described (for exampleBäumlein 1991b).

A second assay permits the quantitative determination of the GUSactivity in the tissue studied. For the quantitative activitydetermination, MUG (4-methylumbelliferyl-β-D-glucuronide) is used assubstrate for β-glucuronidase, and the MUG is cleaved into MU(methylumbelliferone) and glucuronic acid.

To do this, a protein extract of the desired tissue is first preparedand the substrate of GUS is then added to the extract. The substrate canbe measured fluorimetrically only after the GUS has been reacted.Samples which are subsequently measured in a fluorimeter are taken atvarious points in time. This assay may be carried out for example withlinseed embryos at various developmental stages (21, 24 or 30 days afterflowering). To this end, in each case one embryo is ground into a powderin a 2 mL reaction vessel in liquid nitrogen with the aid of a vibrationgrinding mill (Type: Retsch MM 2000). After addition of 100 μL of EGLbuffer (0.1 M KPO₄, pH 7.8; 1 mM EDTA; 5% glycerol; 1 M DTT), themixture is centrifuged for 10 minutes at 25° C. and 14,000×g. Thesupernatant is removed and recentrifuged. Again, the supernatant istransferred to a new reaction vessel and kept on ice until further use.25 μL of this protein extract are treated with 65 μL of EGL buffer(without DTT) and employed in the GUS assay. 10 μL of the substrate MUG(10 mM 4-methylumbelliferyl-β-D-glucuronide) are now added, the mixtureis vortexed, and 30 μL are removed immediately as zero value and treatedwith 470 μL of Stop buffer (0.2 M Na₂CO₃). This procedure is repeatedfor all of the samples at an interval of 30 seconds. The samples takenwere stored in the refrigerator until measured. Further readings weretaken after 1 h and after 2 h. A calibration series which containedconcentrations from 0.1 mM to 10 mM MU (4-methylumbelliferone) wasestablished for the fluorimetric measurement. If the sample values wereoutside these concentrations, less protein extract was employed (10 μL,1 μL, 1 μL from a 1:10 dilution), and shorter intervals were measured (0h, 30 min, 1 h). The measurement was carried out at an excitation of 365nm and an emission of 445 nm in a Fluoroscan II apparatus (Labsystem).As an alternative, the substrate cleavage can be monitoredfluorimetrically under alkaline conditions (excitation at 365 nm,measurement of the emission at 455 nm; Spectro Fluorimeter BMGPolarstar+) as described in Bustos (1989). All the samples weresubjected to a protein concentration determination by the method ofBradford (1976), thus allowing an identification of the promoteractivity and promoter strength in various tissues and plants.

Example 6 Constitutive Expression in Maize 6.1 RiceCCoAMT1-Promoter::Zmubiquitin-Intron::GUS::CCoAMT1 terminator(pBPSMM325)

CCoAMT1 promoter in combination with CCoAMT1 terminator shows strongconstitutive and ubiquitous expression in all tissues and organs atdifferent developmental stages. Strong ubiquitous expression can also bedetected in in vitro plants.

TABLE 4 GUS expression controlled by monocot constitutive promotercandidates Tissues/Developmental Promoter (GUS expression levels) stagespBPSMM232* pBPSMM247* pBPSMM272 pBPSMM325 3 days after co-cultivation++++ +++ ++ +++ Leaves at 5-leaf stage +++++ +++++ ++++ ++++ Roots at5-leaf stage +++++ +++++ ++++ ++++ Leaves at flowering stage +++++ +++++++++ +++ Stem +++ +++ ++ +++ Pre-pollination +++++ +++++ +++ ++ 5 daysafter pollination [DAP] +++++ +++ (7 DAP) ++++ (7 DAP) ND 30 DAP ++++++++++ ++++ ++ Dry seeds ND +++ ND ++ Imbibition/germination +++++ +++++++ ND *Positive controls as a constitutive promoter (pBPSMM232 =Zm.ubiquitin promoter::Zm.ubiquitin intron::GUS (PIV2)::NOS terminator;pBPSMM247 = sugarcane bacilliform virus promoter::GUS (PIV2) ::NOSterminator); a range of GUS expression levels measured by histochemicalassay (− to +++++), ND: not determined yet

Example 7 Root and Kernel Preferable Expression in Maize 7.1 RiceOs.CCoAMT1 Promoter::Zm.Ubiquitin Intron::GUS (PIV2)::NOS Terminator

Caffeoyl-CoA-O-methyltransferase (CCoAMT1)promoter::ubiquitin-intron::NOS terminator (pBPSMM271) showed lowexpression in leaves and stem of T1 plants but strong expression inroots. GUS stain was also detected in kernel and pollen.

7.2 Rice SI Promoter::Zm.Ubiquitin Intron::GUS (PIV2)::NOS Terminator

OsC-8,7-sterol-isomerase promoter::Zm.ubiquitinintron::NOS terminator(pBPSMM331) showed weak expression in most parts of the plants but goodexpression in roots and kernels.

7.3 Maize HRGP Promoter::Zm.Ubiquitin Intron::GUS (PIV2)::HRGPTerminator

HRGP promoter containing the ubiquitin intron and the HRGP terminator(pBPSET003) showed no expression in leaves but strong expression inroots and silk. In kernels expression is predominantly in the embryo andonly weak in the endosperm.

7.4 Maize LDH Promoter::Zm.Ubiquitin Intron::GUS (PIV2)::NOS or LDHTerminator

Lactate-dehydrogenase (LDH) promoter::Zm.ubiquitinintron::NOS or LDHterminator (pBPSMM272 or pBPSET007, respectively) showed weak expressionin leaves but good expression in roots and kernels.

TABLE 5 GUS expression controlled by monocot root and kernel-preferablepromoter candidates Promoter (GUS expression levels) pBPSMM272 Tissues &Developmental or pBP- stages pBPSMM232* pBPSMM271 pBPSMM331 pBPSET003SET007 3 days after co- ++++ + ND ND +++ cultivation Leaves at 5-leaf+++++ + + − ++ stage Roots at 5-leaf +++++ ++++ +++ ++++ ++++ stageLeaves at flowering +++++ + ++ − ++ stage Stem +++ + ND ND +Pre-pollination +++++ +++ ++++ ND +++ 5 days after pollination +++++ +++ND ND +++ [DAP] 30 DAP ++++ +++ ++ ++ +++ Dry seeds ND ND ND ND NDImbibition/ +++++ +++ ND ND +++ germination *positive control as aconstitutive promoter (pBPSMM232 = Zm.ubiquitin promoter::Zm.ubiquitinintron::GUS (PIV2)::NOS terminator); a range of GUS expression levelsmeasured by histochemical assay (− to +++++), ND: not determined yet

Example 8 Leaf and Endosperm Preferable Expression in Maize

Os.CP12 promoter::Zm.ubiquitin intron::GUS (PIV2)::NOS terminator(pBPSMM304) showed strong expression in leaves and endosperm, but not inroots or embryo.

TABLE 6 GUS expression controlled by leaf and endosperm-preferablemonocot promoter Promoter (GUS expression levels) Tissues/Developmentalstages pBPSMM232* pBPSMM304 3 days after co-cultivation ++++ + In vitroleaves +++++ ++++ In vitro roots +++++ − Leaves +++++ ++++ Roots +++++ −Kernel pre-pollination +++++ + Kernel 30 DAP - Endosperm +++++ ++++Kernel 30 DAP - Embryo +++++ − Dry seeds ++++ ND *positive control as aconstitutive promoter (pBPSMM232 = Znn.ubiquitin promoter::Zm.ubiquitinintron::GUS (PIV2)::NOS terminator); a range of GUS expression levelsmeasured by histochemical assay (− to +++++), ND: not determined yet

Example 9 Analysis of Drought-Inducible Expression Using Real Time RTPCR Analysis

Young maize plants were grown from seeds in the greenhouse understandard conditions. When plants had five true leaves (5-leaf stage)water was withheld from the drought-stress plants while wateringcontinued for the water control plants. The 0-timepoint is taken at thelast watering day. After that the first time point is taken when thesoil is dry but plants don't show symptoms of drought yet (approximately3 days), the second time point is taken when at least one leaf of everyplant shows “rolling” (mild symptoms, approximately 5 days) and the lasttime point is taken when all leaves show “rolling” (severe symptoms,approximately 7 days).

Expression levels of the reporter gene was measured at the mRNA levelsusing a GeneAmp 5700 Sequence Detection System (Applied Biosystems).Total nucleic acids were extracted from maize leaf samples taken atvarious time points during the drought stress experiments using theWizard Magnetic 96 DNA plant System kit (Promega, FF3661). Subsequently,DNA was removed from the samples using the DNA-free kit (Ambion, #1906).The resulting DNA-free RNA solution was used in subsequent PCRreactions. The one-batch RT/quantitative PCR reactions for expressionanalysis contained:

  10 μL RNA solution   15 μL SYBR Green master Mix (2X; Eurogentec#RTSNRT032X-1) 0.15 μL reverse transcriptase + inhibitor1 mix(Eurogentec #RTSNRT032X-1)  0.6 μL forward primer (10 pmol/μL)  0.6 μLreverse primer (10 pmol/μL) 3.65 μL sterile water

The PCR program was:

1 cycle 48° C. for 30 min (RT reaction)40 cycles 90° C. for 10 min; 95° C. for 15 sec and 60° C. for 1 min

The amplification was followed by a dissociation protocol:

95° C. for 15 sec, 60° C. for 20 sec20 min slow ramp from 60° C. to 95° C.

TABLE 7 Primer sequences of the GUS gene and controlgene encoding microtubule-associated protein 1 light chain 3 GenePrimers SEQ ID GUS Fwd: 5′-ttacgtggcaaaggattcgat SEQ ID NO: 91Rev: 5′-gccccaatccagtccattaa SEQ ID NO: 92 ControlFwd: 5′-tctgccttgcccttgctt SEQ ID gene NO: 93Rev: 5′-caattgcttggcaggtcttattt SEQ ID NO: 94

For each timepoint of the drought-stress experiment RNA solution of eachsample was used in two qPCR reactions that were run at the same time onthe same plate. One reaction contained the GUS primers the secondreaction contained primers for an endogenous control gene from maize.This endogenous control gene shows stable expression under stressconditions in a variety of tissues.

The preset baseline and threshold of the GeneAmp 5700 software were usedin all experiments to generate raw data (cycle numbers). In order tonormalize the values obtained with the GeneAmp 5700 Sequence DetectionSystem the values of the endogenous primer reactions were subtractedfrom the values of the GUS primer reactions. The difference in cyclenumbers was then used to calculate the change of expression levels. Astemplates are exponentially amplified during PCR one cycle differenceequals a two-fold difference in template levels. Results are shown as anx-fold induction compared to the O-timepoint that is set to 1.

9.1 Drought-Inducible Expression Controlled by Maize LDH Promoter

In T1 plants containing a single copy shows strong constitutiveexpression in roots and in kernels. Expression in leaves and stem atdifferent developmental stages was weak. Upon drought-stress expressionin leaves was induced two-fold compared to well-watered control.

Example 10 Utilization of Transgenic Crops

A reporter gene in pBPSMM325 can be replaced with gene of interest toexpress in a constitutive and ubiquitous manner, a reporter gene inpBPSMM271, pBPSMM331, pBPSET003, and pBPSET007 can be replaced with geneof interest to express mostly in roots and kernel, a reporter gene inpBPSMM304 can be replaced with gene of interest to express mostly inleaves and endosperm (e.g., by antisense or double-stranded RNA),thereby improving—for example—biomass and/or yield, or tolerant tobiotic and abiotic environmental stresses. The chimeric constructs aretransformed into monocotyledonous plants. Standard methods fortransformation in the art can be used if required. Transformed plantsare regenerated using known methods. Various phenotypes are measured todetermine improvement of biomass, yield, fatty acid composition, highoil, disease tolerance, or any other phenotypes that link yieldenhancement or stability. Gene expression levels are determined atdifferent stages of development and at different generations (T₀ to T₂plants or further generations). Results of the evaluation in plants leadto determine appropriate genes in combination with this promoter toincrease yield, improve disease tolerance, and/or improve abiotic stresstolerance.

Example 11 Expression of Selectable Marker Gene in Monocotyledonousplants

A reporter gene in pBPSMM325 can be replaced with a selectable markergene and transformed into monocotyledonous plants such as rice, barley,maize, wheat, or ryegrass but is not restricted to these plant species.Any methods for improving expression in monocotyledonous plants areapplicable such as addition of intron or exon with intron in 5′UTReither non-spliced or spliced. Standard methods for transformation inthe art can be used if required. Transformed plants are selected underthe selection agent of interest and regenerated using known methods.Selection scheme is examined at early developmental stages of tissues ortissue culture cells. Gene expression levels can be determined atdifferent stages of development and at different generations (T₀ to T₂plants or further generations). Results of the evaluation in plants leadto determine appropriate genes in combination with this promoter.

Example 12 Expression of Transgene for Root Vigor in MonocotyledonousPlants

A reporter gene in pBPSMM271, pBPSMM331, pBPSET003, pBPSMM272, andpBPSET007 can be replaced with gene of interest to express mostly inroots, which affects root architecture and transformed intomonocotyledonous plants such as rice, barley, maize, wheat, or ryegrassbut is not restricted to these plant species. Any methods for improvingexpression in monocotyledonous plants are applicable such as addition ofintron or exon with intron in 5′UTR either non-spliced or spliced.Standard methods for transformation in the art can be used if required.Transformed plants are selected under the selection agent of interestand regenerated using known methods. Selection scheme is examined atearly developmental stages of tissues or tissue culture cells. Geneexpression levels can be determined at different stages of developmentand at different generations (T₀ to T₂ plants or further generations).Results of the evaluation in plants lead to determine appropriate genesin combination with this promoter.

Example 13 Expression of Transgene for Feed and Food in MonocotyledonousPlants

A reporter gene in pBPSMM271, pBPSMM331, pBPSET003, pBPSMM272,pBPSET007, and pBPSMM304 can be replaced with gene of interest toexpress mostly in kernel, which improve nutrition in embryo andendosperm and transformed into monocotyledonous plants such as rice,barley, maize, wheat, or ryegrass but is not restricted to these plantspecies. Any methods for improving expression in monocotyledonous plantsare applicable such as addition of intron or exon with intron in 5′UTReither non-spliced or spliced. Standard methods for transformation inthe art can be used if required. Transformed plants are selected underthe selection agent of interest and regenerated using known methods.Selection scheme is examined at early developmental stages of tissues ortissue culture cells. Gene expression levels can be determined atdifferent stages of development and at different generations (T₀ to T₂plants or further generations). Results of the evaluation in plants leadto determine appropriate genes in combination with this promoter.

Example 14 Deletion Analysis

The cloning method is described by Rouster (1997) and Sambrook (1989).Detailed mapping of these promoters (i.e., narrowing down of the nucleicacid segments relevant for its specificity) is performed by generatingvarious reporter gene expression vectors which firstly contain theentire promoter region and secondly various fragments thereof. Firstly,the entire promoter region or fragments thereof are cloned into a binaryvector containing GUS or other reporter gene. To this end, fragments areemployed firstly, which are obtained by using restriction enzymes forthe internal restriction cleavage sites in the full-length promotersequence. Secondly, PCR fragments are employed which are provided withcleavage sites introduced by primers. The chimeric GUS constructscontaining various deleted promoters are transformed into Zea mays,Arabidopsis and other plant species using transformation methods in thecurrent art. Promoter activity is analyzed by using GUS histochemicalassays or other appropriate methods in various tissues and organs at thedifferent developmental stages. Modification of the promoter sequencescan eliminate leakiness based on our needs.

Example 15 In Vivo Mutagenesis

The skilled worker is familiar with a variety of methods for themodification of the promoter activity or identification of importantpromoter elements. One of these methods is based on random mutationfollowed by testing with reporter genes as described above. The in vivomutagenesis of microorganisms can be achieved by passage of the plasmid(or of another vector) DNA through E. coli or other microorganisms (forexample Bacillus spp. or yeasts such as Saccharomyces cerevisiae) inwhich the ability of maintaining the integrity of the geneticinformation is disrupted. Conventional mutator strains have mutations inthe genes for the DNA repair system (for example mutHLS, mutD, mutT andthe like; for reference, see Rupp 1996). The skilled worker is familiarwith these strains. The use of these strains is illustrated for exampleby Greener (1994). The transfer of mutated DNA molecules into plants ispreferably effected after selection and testing of the microorganisms.Transgenic plants are generated and analyzed as described above.

Example 16 PLACE Analysis for Os.CCoAMT1 Promoter (SEQ ID NO: 1)

Based on the below given PLACE results are potential TATA box islocalized at base pair 952 to base pair 958 of SEQ ID NO: 1. Inconsequence the 5′ untranslated region starts at about base pair 993 andextends to base pair 1,035 of SEQ ID NO: 1. The sequence described bySEQ ID NO: 2 ends 17 base pairs before the ATG start codon.

The following clusters of promoter elements were identified in theOs.CCoAMT1 promoter as described by SEQ ID NO: 1:

Position IUPAC from-to Str. Sequence LTRECOREATCOR15 33-39 (−) TCCGACCTATABOX3 45-51 (+) TATTAAT CCAATBOX1 86-90 (−) CCAAT SEF1MOTIF  99-107(+) ATATTTATA TATAPVTRNALEU 101-113 (+) ATTTATATATTAA TATABOX2 101-107(−) TATAAAT TATABOX4 103-109 (−) TATATAA WBOXATNPR1 132-146 (−)ATTGACGTCGAATTG HEXMOTIFTAH3H4 135-147 (+) TTCGACGTCAATA TGACGTVMAMY137-149 (−) TCTATTGACGTCG CGACGOSAMY3 137-141 (+) CGACG ACGTCBOX 138-143(+) GACGTC ACGTCBOX 138-143 (−) GACGTC BOXIINTPATPB 145-150 (+) ATAGAASP8BFIBSP8AIB 169-176 (−) ACTGTGTA CIACADIANLELHC 190-199 (−) CAATAATATCS1FBOXSORPS1L21 221-226 (+) ATGGTA ABRELATERD1 226-238 (+) ATCAACGTGATCGCIACADIANLELHC 228-237 (+) CAACGTGATC BP5OSWX 228-234 (+) CAACGTG MYBST1267-273 (+) GGGATAT ABRELATERD1 307-319 (−) TAAAACGTGTGCT QARBNEXTA310-316 (−) AACGTGT CCAATBOX1 325-329 (+) CCAAT SEF3MOTIFGM 333-338 (+)AACCCA TATABOXOSPAL 360-366 (+) TATTTAA TATABOX2 366-372 (−) TATAAATQELEMENTZMZM13 375-389 (−) CAGGTCACGAATTCA WBOXHVISO1 386-400 (+)CCTGACTCACTCACA GCN4OSGLUB1 387-395 (−) GTGAGTCAG WBOXHVISO1 413-427 (−)GGTGACTGAGACAAA SEBFCONSSTPR10A 414-420 (+) TTGTCTC ARFAT 415-420 (+)TGTCTC IBOXCORENT 449-455 (+) GATAAGG IBOXCORE 456-462 (−) GATAAACMYBST1 458-464 (−) AGGATAA CGACGOSAMY3 471-475 (+) CGACG HEXAMERATH4471-476 (−) CCGTCG IBOXCORE 475-481 (−) GATAACC IBOXCORE 527-533 (+)GATAAAG TAAAGSTKST1 527-533 (+) GATAAAG NTBBF1ARROLB 528-534 (−) ACTTTATMYB2AT 543-553 (+) GTTTTAACTGC PALBOXLPC 576-586 (+) CCTCACCAACCMYBPLANT 579-589 (+) CACCAACCTTC MYBPZM 581-586 (+) CCAACC ARFAT 608-613(+) TGTCTC AGCBOXNPGLB 623-629 (+) AGCCGCC RAV1BAT 653-665 (+)ACGCACCTGGCGG ABRELATERD1 689-701 (+) GAAGACGTGGAGG CCAATBOX1 730-734(+) CCAAT PALBOXAPC 737-742 (+) CCGTCC MYB1AT 768-773 (+) AAACCAPALBOXLPC 774-784 (+) CCTCACCAACC MYBPLANT 777-787 (+) CACCAACCCAAMYBPZM 779-784 (+) CCAACC SEF3MOTIFGM 781-786 (+) AACCCA CAREOSREP1785-790 (+) CAACTC BOXCPSAS1 816-822 (+) CTCCCAC GCBP2ZMGAPC4 831-839(−) GTGGGCCCG RAV1BAT 834-846 (+) GCCCACCTGTCGG DRECRTCOREAT 841-847 (−)GCCGACA CCA1ATLHCB1 880-887 (−) AAAAATCT PYRIMIDINEBOXHVEPB 883-890 (+)TTTTTTCC MYCATERD1 920-926 (−) CATGTGA MYCATRD22 921-927 (+) CACATGCBOXCPSAS1 932-938 (+) CTCCCAC TATAPVTRNALEU 948-960 (−) GTTTATATAGCGCTATABOX4 952-958 (+) TATATAA CGACGOSAMY3 970-974 (−) CGACG DRECRTCOREAT981-987 (−) GCCGACG CGACGOSAMY3 981-985 (−) CGACG WBOXATNPR1 982-996 (−)ATTGACTTCGCCGAC

Example 17 PLACE Analysis for Os.SI Promoter (SEQ ID NO: 6)

Based on the below given PLACE results, no conventional TATA box hasbeen found in this promoter region (797 bp). The following clusters ofpromoter elements were identified in the Zm.SI promoter as described bySEQ ID NO: 6:

Position IUPAC From-to Str. Sequence TBOXATGAPB 66-71 (+) ACTTTG RAV1AAT 96-100 (+) CAACA CATATGGMSAUR 113-118 (+) CATATG CATATGGMSAUR 113-118(−) CATATG −300ELEMENT 127-135 (+) TGCAAAATC POLLEN2LELAT52 159-167 (−)TCCACCATA CGACGOSAMY3 259-263 (+) CGACG HEXAMERATH4 259-264 (−) CCGTCGCGACGOSAMY3 288-292 (+) CGACG HEXAMERATH4 288-293 (−) CCGTCG GCCCORE384-390 (−) CGCCGCC GCCCORE 387-393 (−) CGCCGCC GCCCORE 390-396 (−)TGCCGCC GRAZMRAB28 390-398 (−) CATGCCGCC DRECRTCOREAT 397-403 (−)GCCGACA GCCCORE 401-407 (−) CGCCGCC BS1EGCCR 422-427 (+) AGCGGGCGACGOSAMY3 434-438 (+) CGACG HEXAMERATH4 434-439 (−) CCGTCG GCCCORE450-456 (−) CGCCGCC DRECRTCOREAT 456-462 (−) GCCGACC MYBPZM 552-557 (−)CCAACC CGACGOSAMY3 559-563 (+) CGACG SEF3MOTIFGM 593-598 (−) AACCCALRENPCABE 607-619 (−) GCCGACGTGGCAT ABREOSRAB21 608-620 (+)TGCCACGTCGGCC DRECRTCOREAT 613-619 (−) GCCGACG CGACGOSAMY3 613-617 (−)CGACG TAAAGSTKST1 634-640 (−) GTTAAAG MYBGAHV 637-643 (+) TAACAAAQELEMENTZMZM13 672-686 (−) TAGGTCAATGCCTCA ELRECOREPCRP1 678-692 (+)ATTGACCTACCTTGG MYBPZM 683-688 (+) CCTACC LTRECOREATCOR15 733-739 (+)CCCGACG CGACGOSAMY3 735-739 (+) CGACG CCAATBOX1 756-760 (+) CCAAT

Example 18 PLACE Analysis for Zm.HRGP Promoter (SEQ ID NO: 11)

Based on the below given PLACE results are potential TATA box islocalized at base pair 1,071 to base pair 1,077 of SEQ ID NO: 11. Inconsequence the 5′ untranslated region starts at about base pair 1,112and extends to base pair 1,182 of SEQ ID NO: 11. The sequence describedby SEQ ID NO: 11 end just before the ATG start codon.

The following clusters of promoter elements were identified in theZm.HRGP promoter as described by SEQ ID NO: 11:

Position IUPAC from-to Str. Sequence ACGTCBOX 30-35 (+) GACGTC ACGTCBOX30-35 (−) GACGTC CGACGOSAMY3 32-36 (−) CGACG BOXIINTPATPB  95-100 (+)ATAGAA CGACGOSAMY3 138-142 (+) CGACG HEXAMERATH4 138-143 (−) CCGTCGMYBPZM 146-151 (+) CCAACC GT1CORE 167-177 (+) CGGTTAAATAG TATABOXOSPAL170-176 (−) TATTTAA CGACGOSAMY3 179-183 (+) CGACG HEXAMERATH4 179-184(−) CCGTCG MYCATERD1 223-229 (−) CATGTGC MYCATRD22 224-230 (+) CACATGCNTBBF1ARROLB 238-244 (+) ACTTTAT TAAAGSTKST1 239-245 (−) TATAAAGTATABOX2 242-248 (+) TATAAAT IBOXCORE 276-282 (−) GATAATA REBETALGLHCB21291-297 (+) CGGATAG IBOXCORE 296-302 (−) GATAACT IBOXCORE 325-331 (+)GATAACT NTBBF1ARROLB 329-335 (+) ACTTTAT TAAAGSTKST1 330-336 (−) TATAAAGTATABOX2 333-339 (+) TATAAAT GCCCORE 358-364 (−) TGCCGCC ABRELATERD1366-378 (−) GCAGACGTGTGCG BOXIINTPATPB 409-414 (+) ATAGAALTRECOREATCOR15 429-435 (+) TCCGACC ASF1MOTIFCAMV 447-459 (+)GACATTGACGGAT WBOXATNPR1 450-464 (+) ATTGACGGATCCAGA ELRECOREPCRP1466-480 (−) TTTGACCGGATCGCC CGACGOSAMY3 485-489 (+) CGACG RAV1AAT537-541 (−) CAACA TATABOX2 560-566 (+) TATAAAT IBOXCORE 595-601 (−)GATAATA IBOXCORE 615-621 (−) GATAACG SV40COREENHAN 643-650 (−) GTGGATCGABRELATERD1 644-656 (−) AACGACGTGGATC CGACGOSAMY3 650-654 (−) CGACGMYCATERD1 672-678 (−) CATGTGC MYCATRD22 673-679 (+) CACATGG AGCBOXNPGLB678-684 (−) AGCCGCC DPBFCOREDCDC3 688-694 (−) ACACAAG ABRELATERD1744-756 (+) AATAACGTGAGTA RAV1BAT 791-803 (+) ATCCACCTGCTCC MYBST1823-829 (−) TGGATAG AMYBOX2 824-830 (+) TATCCAT TATCCAOSAMY 824-830 (+)TATCCAT WBOXATNPR1 829-843 (−) GTTGACGAATGGAAT ASF1MOTIFCAMV 834-846 (−)CATGTTGACGAAT RAV1AAT 840-844 (+) CAACA RYREPEATBNNAPA 841-851 (+)AACATGCAGGT INTRONLOWER 845-850 (+) TGCAGG CCAATBOX1 895-899 (+) CCAATMYCATERD1 944-950 (−) CATGTGG MYCATRD22 945-951 (+) CACATGG HEXAMERATH41006-1011 (+) CCGTCG CGACGOSAMY3 1007-1011 (−) CGACG ASF1MOTIFCAMV1014-1026 (−) TCTCGTGACGCCC TATABOX4 1071-1077 (+) TATATAA CCAATBOX11121-1125 (−) CCAAT MYBPZM 1136-1141 (+) CCAACC MYB2AT 1152-1162 (−)TCAGTAACTGC

Example 19 PLACE Analysis for Zm.LDH Promoter (SEQ ID NO: 19)

Based on the below given PLACE results are potential TATA box islocalized at base pair 906 to base pair 912 of SEQ ID NO: 19. Inconsequence the 5′ untranslated region starts at bout base pair 947 andextends to base pair 1,060 of SEQ ID NO: 19. The sequence described bySEQ ID NO: 19 ends 31 base pairs before the ATG start codon.

The following clusters of promoter elements were identified in theZm.LDH promoter as described by SEQ ID NO: 19.

Position IUPAC from-to Str. Sequence TATABOX4 15-21 (−) TATATAA5256BOXLELAT5256 16-27 (−) TGTGGTTATATA TATABOX4 16-22 (+) TATATAAMYB1AT 20-25 (+) TAACCA ASF1MOTIFCAMV 39-51 (−) AATAATGACGCAG MYB1AT93-98 (+) AAACCA AACACOREOSGLUB1 114-120 (−) AACAAAC LTRE1HVBLT49119-124 (−) CCGAAA REALPHALGLHCB21 144-154 (−) AACCAACGATA WBOXHVISO1172-186 (−) GATGACTCGTACGGC IBOXCORE 201-207 (−) GATAAAA DRE2COREZMRAB17208-214 (+) ACCGACT RAV1AAT 238-242 (+) CAACA ASF1MOTIFCAMV 254-266 (−)GTTCGTGACGCTT TAAAGSTKST1 339-345 (+) ATTAAAG NTBBF1ARROLB 340-346 (−)ACTTTAA RAV1AAT 361-365 (+) CAACA CATATGGMSAUR 378-383 (+) CATATGCATATGGMSAUR 378-383 (−) CATATG −10PEHVPSBD 397-402 (−) TATTCTTAAAGSTKST1 419-425 (+) GTTAAAG RAV1AAT 435-439 (−) CAACA CAREOSREP1448-453 (−) CAACTC CGACGOSAMY3 456-460 (+) CGACG IBOXCORE 476-482 (+)GATAAAA TBOXATGAPB 489-494 (−) ACTTTG IBOXCORE 561-567 (+) GATAAAATATABOXOSPAL 574-580 (+) TATTTAA TATABOXOSPAL 583-589 (−) TATTTAAGT1CORE 597-607 (−) AGGTTAAAACT S1FBOXSORPS1L21 667-672 (+) ATGGTAPROLAMINBOXOSGLUB1 678-686 (+) TGCAAAGAG MYB2AT 732-742 (+) TGGGTAACTGTWBOXATNPR1 735-749 (−) GTTGACGACAGTTAC ASF1MOTIFCAMV 740-752 (−)CCGGTTGACGACA CCA1ATLHCB1 810-817 (−) AAAAATCT GT1CORE 831-841 (−)TGGTTAAAATT MYB1AT 836-841 (+) TAACCA SV40COREENHAN 861-868 (+) GTGGTTTGMYB1AT 862-867 (−) AAACCA TATABOX3 890-896 (+) TATTAAT WUSATAg 892-898(+) TTAATGG TATAPVTRNALEU 902-914 (−) CTTTATATATTCA TATABOX4 906-912 (+)TATATAA TAAAGSTKST1 908-914 (+) TATAAAG BOXCPSAS1 937-943 (+) CTCCCACOCTAMERMOTIFTAH3H4  998-1005 (−) CGCGGATC ELRECOREPCRP1 1010-1024 (+)TTTGACCCAACCAGA MYBPZM 1016-1021 (+) CCAACC CIACADIANLELHC 1017-1026 (+)CAACCAGATC ABRELATERD1 1031-1043 (−) GATTACGTGTGTG DPBFCOREDCDC31032-1038 (+) ACACACG

Example 20 PLACE Analysis for Os.Cp12 Promoter (SEQ ID NO: 27)

Based on the below given PLACE results are potential TATA box islocalized at base pair 908 to base pair 914 of SEQ ID NO:27. Inconsequence the 5′ untranslated region starts at about base pair 960 andextends to base pair 998 of SEQ ID NO:27.

The following clusters of promoter elements were identified in theOs.Cp12 promoter as described by SEQ ID NO:27:

Position IUPAC from-to Str. Sequence AMMORESIIUDCRNIA1 28-35 (+)GGAAGGGT ABRELATERD1 57-69 (+) GAGGACGTGAGGC LTRE1HVBLT49 88-93 (−)CCGAAA −300ELEMENT  96-104 (+) TGAAAAATT SEF1MOTIF 108-116 (−) ATATTTAAATATABOXOSPAL 109-115 (−) TATTTAA WBOXATNPR1 173-187 (−) GTTGACTGGGCCTTAMYB2AT 213-223 (+) GCTGTAACTGG RAV1AAT 244-248 (−) CAACA CIACADIANLELHC286-295 (+) CAAGGCCATC MYB1AT 296-301 (−) AAACCA SV40COREENHAN 310-317(−) GTGGTAAG CCAATBOX1 352-356 (+) CCAAT −300ELEMENT 362-370 (−)TGTAAAGTT NTBBF1ARROLB 363-369 (+) ACTTTAC −300CORE 364-370 (−) TGTAAAGTAAAGSTKST1 364-370 (−) TGTAAAG CCAATBOX1 392-396 (−) CCAAT −300ELEMENT403-411 (+) TGAAAAATA RAV1BAT 443-455 (+) CTGCACCTGTACA MYBPLANT 519-529(+) CACCAAACTTT EVENINGAT 543-551 (−) AAAATATCT LTRE1HVBLT49 557-562 (+)CCGAAA RAV1AAT 620-624 (+) CAACA MYBST1 645-651 (−) TGGATAC TATCCAOSAMY646-652 (+) TATCCAA MYBPZM 649-654 (+) CCAACC WBOXHVISO1 676-690 (−)GATGACTGTGGGTGT ELRECOREPCRP1 698-712 (−) TTTGACCGTGAAAAC ABREAZMRAB28807-819 (−) TGCCACGTGGGCT GBOXLERBCS 808-820 (+) GCCCACGTGGCACUPRMOTIFIIAT 813-831 (−) CCAATCGTCGTGTGCCACG CGACGOSAMY3 822-826 (+)CGACG CCAATBOX1 827-831 (−) CCAAT IBOXCORENT 842-848 (+) GATAAGAABRELATERD1 853-865 (−) GACGACGTGCACT CGACGOSAMY3 859-863 (−) CGACGCGACGOSAMY3 862-866 (−) CGACG UPRMOTIFIIAT 879-897 (+)CCTTCTCCCCCACCCCACG TATAPVTRNALEU 904-916 (−) GTTTATATATATA TATABOX4908-914 (+) TATATAA CGACGOSAMY3 948-952 (−) CGACG RAV1AAT 953-957 (+)CAACA RAV1AAT 994-998 (+) CAACA

Example 21 Vector Construction for Overexpression and Gene “Knockout”Experiments 21.1 Overexpression

Vectors used for expression of full-length “candidate genes” of interestin plants (overexpression) are designed to overexpress the protein ofinterest and are of two general types, biolistic and binary, dependingon the plant transformation method to be used.

For biolistic transformation (biolistic vectors), the requirements areas follows:

-   1. a backbone with a bacterial selectable marker (typically, an    antibiotic resistance gene) and origin of replication functional in    Escherichia coli (E. coli; e.g., ColE1), and-   2. a plant-specific portion consisting of:    -   a. a gene expression cassette consisting of a promoter (e.g.        ZmUBlint MOD), the gene of interest (typically, a full-length        cDNA) and a transcriptional terminator (e.g., Agrobacterium        tumefaciens nos terminator);    -   b. a plant selectable marker cassette, consisting of a suitable        promoter, selectable marker gene (e.g., D-amino acid oxidase;        dao1) and transcriptional terminator (eg. nos terminator).

Vectors designed for transformation by Agrobacterium tumefaciens (A.tumefaciens; binary vectors) consist of:

-   1. a backbone with a bacterial selectable marker functional in    both E. coli and A. tumefaciens (e.g., spectinomycin resistance    mediated by the aadA gene) and two origins of replication,    functional in each of aforementioned bacterial hosts, plus the A.    tumefaciens virG gene;-   2. a plant-specific portion as described for biolistic vectors    above, except in this instance this portion is flanked by A.    tumefaciens right and left border sequences which mediate transfer    of the DNA flanked by these two sequences to the plant.

21.2 Gene Silencing Vectors

Vectors designed for reducing or abolishing expression of a single geneor of a family or related genes (gene silencing vectors) are also of twogeneral types corresponding to the methodology used to downregulate geneexpression: antisense or double-stranded RNA interference (dsRNAi).

(a) Anti-sense

For antisense vectors, a full-length or partial gene fragment(typically, a portion of the cDNA) can be used in the same vectorsdescribed for full-length expression, as part of the gene expressioncassette. For antisense-mediated down-regulation of gene expression, thecoding region of the gene or gene fragment will be in the oppositeorientation relative to the promoter; thus, mRNA will be made from thenon-coding (antisense) strand in planta.

(b) dsRNAi

For dsRNAi vectors, a partial gene fragment (typically, 300 to 500 basepairs long) is used in the gene expression cassette, and is expressed inboth the sense and anti-sense orientations, separated by a spacer region(typically, a plant intron, e.g. the OsSH1 intron 1, or a selectablemarker, e.g. conferring kanamycin resistance). Vectors of this type aredesigned to form a double-stranded mRNA stem, resulting from thebasepairing of the two complementary gene fragments in planta.

Biolistic or binary vectors designed for overexpression or knockout canvary in a number of different ways, including e.g. the selectablemarkers used in plant and bacteria, the transcriptional terminators usedin the gene expression and plant selectable marker cassettes, and themethodologies used for cloning in gene or gene fragments of interest(typically, conventional restriction enzyme-mediated or Gateway™recombinase-based cloning).

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

1. An expression cassette for regulating expression in amonocotyledonous plant, said expression cassette comprising: a) at leastone transcription regulating nucleotide sequence functional in amonocotyledonous plant comprising a sequence selected from the groupconsisting of: i) the nucleotide sequence of SEQ ID NO: 2, 3, 6, 7, 8,11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58,61, 62, 63, 66, 67, 68, 71, 72, or 73; ii) a fragment of at least 50consecutive bases of the nucleotide sequence of SEQ ID NO: 2, 3, 6, 7,8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57,58, 61, 62, 63, 66, 67, 68, 71, 72, or 73; iii) a nucleotide sequencehaving at least 90% sequence identity to the nucleotide sequence of SEQID NO: 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24,27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73; and iv) anucleotide sequence capable of hybridizing to the nucleotide sequence ofi) or the complement thereof in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C.;and b) at least one nucleic acid sequence which is heterologous inrelation to said transcription regulating nucleotide sequence.
 2. Theexpression cassette of claim 1, wherein expression of the nucleic acidsequence of b) results in expression of a protein, an antisense RNA, asense RNA, or a double-stranded RNA.
 3. The expression cassette of claim1, wherein expression of the nucleic acid sequence of b) in a plantconfers an agronomically valuable trait to said plant.
 4. The expressioncassette of claim 1, further comprising at least one element selectedfrom the group consisting of: a) a 5′-untranslated region of a plantexpressed gene; b) an intron sequence from a plant expressed gene; andc) a transcription termination sequence from a plant expressed gene. 5.The expression cassette of claim 4, wherein the transcriptiontermination sequence of c) comprises the nucleotide sequence of SEQ IDNO: 32, 34, or
 35. 6. The expression cassette of claim 4, wherein theintron sequence of b) has expression enhancing properties.
 7. Theexpression cassette of claim 4, wherein the intron sequence of b) is anintron from an ubiquitin, actin, or alcohol dehydrogenase gene.
 8. Avector comprising the expression cassette of claim
 1. 9. A transgenichost cell or non-human organism comprising: a) the expression cassetteof claim 1; or b) a vector comprising the expression cassette of a). 10.A cell culture, part, organ, tissue or transgenic propagation materialderived from the non-human organism of claim 9, wherein said cellculture, part, organ, tissue or transgenic propagation materialcomprises the expression cassette.
 11. A method for the transgenicexpression of a nucleic acid, said method comprising growing orculturing the non-human organism of claim 9 or cell cultures, parts,organs, tissues or transgenic propagation material derived therefrom,wherein said cell cultures, parts, organs, tissues or transgenicpropagation material comprise the expression cassette.
 12. A transgenicplant comprising: a) the expression cassette of claim 1; or b) a vectorcomprising the expression cassette of a).
 13. A method for providing atransgenic expression cassette for heterologous expression in amonocotyledonous plant, said method comprising: a) isolating atranscription regulating nucleotide sequence from a monocotyledonousplant, wherein the transcription regulating nucleotide sequence sharesat least 90% sequence identity to the nucleotide sequence of SEQ ID NO:2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 24, 27, 28,29, 56, 57, 58, 61, 62, 63, 66, 67, 68, 71, 72, or 73; and b)functionally linking said transcription regulating nucleotide sequenceto another nucleotide sequence of interest, which is heterologous inrelation to said transcription regulating nucleotide sequence.
 14. Themethod of claim 13, further comprising functionally linking said anothernucleotide sequence of interest to a transcription termination sequence.15. The method of claim 14, wherein said transcription terminationsequence comprises the nucleotide sequence of SEQ ID NO: 32, 34, or 35.16. A method for identifying and/or isolating a sequence withtranscription regulating activity, said method comprising: a) obtainingpolynucleotide sequences sharing at least 90% sequence identity with thenucleotide sequence of SEQ ID NO: 2, 3, 6, 7, 8, 11, 12, 13, 14, 15, 16,19, 20, 21, 22, 23, 24, 27, 28, 29, 56, 57, 58, 61, 62, 63, 66, 67, 68,71, 72, or 73; b) preparing expression cassettes comprising any of thepolynucleotide sequences of a) operably linked to a reporter gene ormarker and optionally a terminator sequence; c) testing the expressioncassettes for expression activity of the polynucleotide sequencecomprised therein; and d) identifying and/or isolating a polynucleotidesequence having transcription regulating activity.
 17. A method fordirecting expression in a monocotyledonous plant, said methodcomprising: a) introducing into a plant cell the expression cassette ofclaim 1; b) selecting a transgenic cell which comprises said expressioncassette, and c) regenerating a plant from the transgenic cell, whereinthe transcription regulating nucleotide sequence directs expression ofthe nucleic acid sequence which is heterologous in relation to saidtranscription regulating nucleotide sequence in the plant.